CN104955959A - Novel compositions, methods and kits for enhancing PCR specificity - Google Patents
Novel compositions, methods and kits for enhancing PCR specificity Download PDFInfo
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Abstract
The present disclosure provides novel primers and a method for the detection of specific nucleic acid sequences. The primers and methods provided herein are useful in a wide variety of molecular biology applications and are particularly useful in allele-specific PCR.
Description
related application
The application requires according to 35U.S.C.119 (e) U.S. Provisional Patent Application the 61/740th that on December 20th, 2012 submits to, the rights and interests of No. 242, with require the U.S. Provisional Patent Application the 61/721st that on November 2nd, 2012 submits to according to 35U.S.C.119 (e), the rights and interests of No. 968, each mode quoted in full at this in described application is incorporated herein.
Technical field
The present invention relates generally to biology field.Specifically, the present invention relates to and be applicable to detect and distinguish the novel primers of nucleic acid.
Background technology
The existence of the specific nucleic acid molecule in sample can be detected and to carry out determining quantitative analysis to it quite important in medical jurisprudence, medical science, epidemiology and public health.Described analysis may be used for the cause of disease such as differentiating transmissible disease, and prediction is individual will suffer from the possibility of genetic diseases, measure the purity of tap water or milk, or differentiate tissue sample.The hope of the effectiveness and suitability that improve described analysis is obstructed because of sensitivity for analysis usually.Therefore, wish very much to develop sensitiveer detection analysis.
Nucleic acid detection assay can such as, based on any feature of nucleic acid molecule, its size, sequence, and if be DNA, and so can also based on the susceptibility digested by restriction endonuclease.The sensitivity of described analysis can be improved by the mode changed to viewer's report or signaling detected result.Therefore, for example, sensitivity for analysis can be improved by using the reagent that can mark with detecting.For this purpose, diversified described mark has been employed.Detectable label comprises such as radio isotope, fluorescent mark, chemiluminescent labeling, bioluminescence marker and enzyme labelling.
Although use the reagent of height detectable label can improve the sensitivity of nucleic acid detection assay, but the sensitivity of described analysis is still subject to the restriction of some factors, described factor includes, but is not limited to nonspecific reaction, and described nonspecific reaction adds background signal and distinguishes the restriction of the sequence differing one or several Nucleotide.For these problems, the various detection and quantivative approach that use DNA cloning are have developed.
Use primer pair by polymerase chain reaction (PCR) DNA amplification, the sequence of described primer determines the specificity of amplification.But the contribution of the amplicon sequence pair specific amplification beyond trigger area is few or without contribution.In typical PCR reaction, use forward and the preferential target of reverse primer and the associated dna sequence that increases.?
in analysis, additionally provide the probe chosen in amplicon sequence and optionally detect and monitor amplified production.Rest part in target sequence beyond primer and probe region does not adopt.Still the specificity improving amplified reaction is needed, especially for the amplification of the sample of one or several Nucleotide of difference.The example of described analysis includes, but is not limited to gene type, Rare allele detects and the pre-amplification of mutant sequence.
Summary of the invention
Provide the composition for detecting target nucleic acid molecules, method and test kit herein.
On the one hand, oligonucleotide (being called " STAR (desired specificities amplification restriction) primer " or " * primer " herein) is provided, it comprises (i) sequence label, be called " STAR sequence label " and (ii) a certain sequence herein, described sequence and target nucleic acid are hybridized and primer extends from it, copy targeting nucleic acid, produces extension products.The partial complementarity of the extension products 3' of STAR sequence label and STAR primer, the complementary portion of extension products is referred to as STAR labeled targets district in this article.When STAR primer hybridization is to target nucleic acid and when extending, extension products is included in the complementary sequence (that is, STAR labeled targets district) of the STAR sequence label of 5' end and the STAR sequence label in the 3' district of extension products.Therefore, STAR primer extension product can inflection self-annealing, forms stem-ring structure thus.
In certain embodiments, STAR sequence label is at the 5' end of STAR primer or near the 5' of STAR primer holds.In certain embodiments, STAR sequence label and all or part of complementation for the binding site of another primer sequence of the extension products that increases in amplified reaction.
On the one hand, providing package is containing the composition of STAR primer.In certain embodiments, providing package contains the composition of the first Oligonucleolide primers and the second Oligonucleolide primers, and wherein the first primer comprises all or part of the identical STAR sequence label with the target nucleic acid hybridization sequences of the second primer.In certain embodiments, the first and second primers are forward and reverse amplimer pair.
In certain embodiments, provide forward and reverse amplimer pair, wherein described centering at least one in comprise all or part of the identical STAR sequence label with the target nucleic acid hybridization sequences of another primer.In certain embodiments, described two right primers all comprise STAR sequence label.
On the one hand, polynucleotide extension products is provided, it is included in the sequence of the STAR sequence label of 5' end and the STAR sequence label complementation with the 3' side of STAR sequence label, and wherein said STAR sequence label and described complementary sequence make extension products can form stem-ring structure.
On the other hand, STAR primer can be used as and strengthen the specific general instrument easily again of PCR.
In certain embodiments, be provided for the method for the improper amplification suppressing or reduce in fact target nucleic acid, described method comprises makes target nucleic acid contact with one or more STAR primer, and wherein said STAR primer comprises 5'STAR sequence label; And extend STAR primer to form extension products, extension products suppresses or substantially reduces the improper amplification of target nucleic acid whereby.
In certain embodiments, be provided for the method detecting one or more target nucleic acid molecules, described method comprises makes one or more target nucleic acid molecules and one or more STAR primer hybridization, and wherein said STAR primer comprises 5'STAR sequence label; Extend STAR primer to form one or more extension products; Amplification extension products is to form one or more amplified production; And detect at least one amplified production of presence or absence, detect one or more target nucleic acid molecules thus.
In certain embodiments, be provided for the method detecting one or more target nucleic acid molecules, described method comprises makes one or more target nucleic acid molecules and one or more STAR primer hybridization, and wherein said STAR primer comprises 5'STAR sequence label; Extend STAR primer to form one or more extension products; Increase extension products when Existing detector probe to form one or more amplified production, and wherein said detector probe comprises at least one Nucleotide of STAR primer; And detect at least one amplified production of presence or absence, detect one or more target nucleic acid molecules thus.
In certain embodiments, be provided for distinguishing the method methylated with non-methylated targets nucleic acid.In certain embodiments, the method detecting two or more different target nucleic acid from single hybridization is provided for.In certain embodiments, be provided for distinguishing two not homoallelic methods in target nucleic acid sample.
In certain embodiments, provide composition, wherein said composition comprises one or more nucleic acid molecule and at least one oligonucleotide, and wherein said oligonucleotide comprises STAR sequence label and wherein said oligonucleotide is oligonucleotide of the present invention.In certain embodiments, described composition comprises nucleic acid polymerase further.In certain embodiments, described composition comprises primer forward or backwards further.In certain embodiments, described composition comprises detector probe further.
In certain embodiments, provide test kit, described test kit may be used for using described oligonucleotide to carry out hybridizing, extending and amplified reaction herein.In certain embodiments, be provided for the test kit detecting or measure nucleic acid synthesis or amplified production, described test kit comprises one or more oligonucleotide disclosed herein, comprises STAR primer.
Provide these and other feature originally teaching content herein.
Accompanying drawing explanation
Fig. 1 schematically describes the design of the STAR primer according to some embodiment disclosed herein.
Fig. 2 schematically describes alternative designs and the application of STAR primer: Fig. 2 A describes substituting STAR design of primers; Fig. 2 B is depicted in DNA methylation assay and uses STAR primer; And Fig. 2 C describes to use STAR primer general probe to carry out gene type.
Fig. 3 schematically describes the STAR primer from the primer binding site on target sequence with different overlap length.
Fig. 4 represents the amplification containment of STAR primer pair target sequence and the sequence of missing the target with different overlap length to graphically: contain target sequence (lineae trapezoidea), the sequence of missing the target (square line) containing PM STAR label, the target sequence (trilateral line) containing mispairing (MM) STAR label and the sequence of missing the target (round wire) containing MM STAR label of mating (PM) STAR label completely.
Fig. 5 schematically describes the next generation
the embodiment of miRNA analytical work stream, employs 5' with 3' and is connected adapter (or connexon), 5' and 3' connecting cleat and STAR primer.
Fig. 6 represents compared to contrast primer to graphically, and when using STAR primer, adapter (connexon) amplification reduces.
Fig. 7 represents to graphically compared to contrast primer, and the sensitivity using STAR primer that the qPCR of target miRNA is detected increases.
Fig. 8 schematically describes the analysis for distinguishing mankind let-7c and let-7b miRNA, employs standard forward primer and employs STAR forward primer.
Embodiment
Provide the composition for detecting target nucleic acid, method and test kit herein.
Need primer pair by pcr amplified dna, the sequence of described primer determines the specificity of amplification.But, in the process constructing primer or probe sequence, be typically not used in the amplicon sequence beyond trigger area.When primer specificity has in limited time, especially when wanted target and unwanted target be only its internal sequence different time, both all can pass through identical primer pair amplifies.Therefore, need to adopt Internal Amplification subsequence to distinguish further.For this purpose, design and test sequence target amplification restricted (STAR) primer disclosed herein, for the wanted target synchronous formation suppressing unwanted amplified production simultaneously of optionally increasing.
STAR primer tasteless nucleotide (also referred to as " * primer ") comprises (i) containing the part of STAR sequence label and (ii) part containing a certain sequence, described sequence and target nucleic acid are hybridized and primer extends from it, copy targeting nucleic acid, produces extension products.The partial complementarity of the extension products 3' of STAR sequence label and STAR primer, the complementary portion of extension products is referred to as STAR labeled targets district in this article.When STAR primer hybridization is to target nucleic acid and when extending, extension products is included in the complementary sequence (that is, STAR labeled targets district) of the STAR sequence label of 5' end and the STAR sequence label in the 3' district of extension products.Therefore, STAR primer extension product can inflection self-annealing, forms stem-ring structure thus.STAR primer is designed to have the 5'STAR sequence label that can form stem-ring structure in extension products and can for any amplicon internal sequence design STAR sequence label of guiding region, probe region or extension products, for challenging application provides excellent design flexibility.
In certain embodiments, STAR sequence label is at the 5' end of STAR primer or near the 5' of STAR primer holds.In certain embodiments, not extend recognition sequence with the target of STAR primer overlapping for STAR sequence label.In certain embodiments, partly, but it is overlapping to extend recognition sequence with the target of STAR primer by halves for STAR sequence label.Target extend recognition sequence refer in STAR primer hybridize to target nucleic acid and primer from its extend with the part forming extension products.In certain embodiments, target extension recognition sequence has specificity to target nucleic acid.In certain embodiments, it is for universal sequence that target extends recognition sequence, such as, in the common adapter being connected to target nucleic acid end.In certain embodiments, target extension recognition sequence comprises poly (T) sequence, for such as hybridizing to polyadenylation target nucleic acid.
In certain embodiments, STAR sequence label and all or part of complementation (referring to such as Fig. 1) for the binding site of another primer sequence of the extension products that increases in amplified reaction.When STAR primer is extended, extension products can folded back on itself, forms stem-ring structure between the complementary sequence of the STAR sequence label thus in the STAR sequence label at 5' end place and the 3' district of extension products of extension products.Stem-the ring structure formed by STAR primer extension product does not comprise the annealing of another primer for the target molecule that increases, and thereby inhibiting the amplification of unwanted target.
In certain embodiments, STAR sequence label and the complementation of a part of amplicon internal sequence.In certain embodiments, STAR sequence label and a part of inner amplicon complementary and complementary with the binding site of another primer sequence.Stem-the ring structure formed by the extension products of this STAR primer has been blocked the annealing of another primer or has been extended by archaeal dna polymerase, has blocked any further amplification of extension products thus.
In certain embodiments, the forward primer in amplified reaction is STAR primer.In certain embodiments, the reverse primer in amplified reaction is STAR primer.In certain embodiments, the forward in amplified reaction and reverse primer are all STAR primers.
In certain embodiments, provide a kind of STAR primer, it comprises the STAR sequence label identical with the sequence in the nucleic acid of described STAR primer institute target.For example, in certain embodiments, STAR primer comprises the STAR sequence label identical with the primer hybridization sequence in the nucleic acid of institute's target.In certain embodiments, STAR primer comprises the STAR sequence label identical with a part for the nucleic acid of institute target, and described part is the 3' of primer hybridization sequence in the nucleic acid of institute's target.In certain embodiments, STAR primer comprises all or part of the STAR sequence label identical with a part for the nucleic acid of institute target with primer hybridization sequence, and described part is the 3' of primer hybridization sequence in the nucleic acid of institute's target.In the described embodiment, the extension products formed through another primer hybridization sequence of target nucleic acid by the STAR primer extension that hybridizes to target nucleic acid has two complementary sequence parts, and it allows extension products to form stem-ring structure.
In certain embodiments, provide a kind of polynucleotide extension products, it is included in the STAR primer of 5' end and described complementary sequence can be used with the sequence of the STAR sequence label complementation of the STAR primer 3' of STAR primer to form stem-ring structure to make extension products.In certain embodiments, the extension products based on STAR primer can form stem-ring structure under PCR elongating temperature.In certain embodiments, the annealing of different primers to extension products is not comprised based on the extension products of STAR primer in stem-ring structure.
In certain embodiments, STAR primer can be designed to comprise 5'STAR sequence label, and it can form stem-ring structure (referring to Fig. 2 A) after primer extension under PCR elongating temperature.The STAR sequence label completely or partially overlapping with another primer for the target molecule that increases can be selected to get rid of the annealing of another primer.Do not wish to fetter by particular theory, seem stem-ring formation than primer annealing to target molecule fast and due to entropy favourable, so stem-ring structure is more stable.Therefore, STAR primer prevents annealing and the extension of another primer effectively, impels PCR efficiency greatly to reduce.
In certain embodiments, provide STAR primer tasteless nucleotide, wherein STAR sequence label is designed to winding in PCR and extends (referring to such as Fig. 2 A).In certain embodiments, the 5' least significant end sequence in STAR primer is designed in the same chain for the initiation structural domain of particular sequence (" x " referring in primer in Fig. 2 A and amplicon) contained in target amplicon.Take turns in PCR second, described sequence can from another primer extension, whereby its inflection and when mating completely self-replacation 5' sequence, make extension products be not useable for amplification thus.The 5' end of STAR primer can be designed to identical with target for containing amplification.Containing normally increasing with the target nucleic acid of STAR sequence label mispairing.
In certain embodiments, provide a kind of composition, it comprises the first and second primers, and wherein the first primer comprises all or part of the identical STAR sequence label with the target binding sequence of the second primer.In certain embodiments, the length of the identical sequence shared between the first and second primers in composition is between about 3 and about 15 Nucleotide.In certain embodiments, the length sharing sequence is 3,4,5,6,7,8,9,10,11,12,13,14 or 15 Nucleotide.
In certain embodiments, provide amplimer pair, comprise STAR sequence label at least one wherein in described primer, wherein STAR sequence label comprises all or part of of the target binding sequence of another primer.In certain embodiments, the primer comprising STAR sequence label is forward primer and another primer is reverse primer.In certain embodiments, the primer comprising STAR sequence label is reverse primer and another primer is forward primer.In certain embodiments, two primers that amplimer is right all comprise STAR sequence label.
In certain embodiments, STAR primer length is between an about 20-60 Nucleotide.In certain embodiments, the length of STAR primer is about 20 to about 50 Nucleotide.In certain embodiments, the length of STAR primer is about 20 to about 45, about 25 to about 50, about 25 to about 45 or about 30 to about 40 Nucleotide.In certain embodiments, the length of STAR primer is about 30,31,32,33,34,35,36,37,38,39,40,41,42,43,44 or 45 Nucleotide.
In certain embodiments, the length of STAR sequence label is about 10 to about 40 Nucleotide.In certain embodiments, STAR sequence label length is between an about 30-35 Nucleotide.In certain embodiments, the length of STAR sequence label is about 10 to about 30, about 10 to about 35, about 10 to about 20, about 15 to about 30, about 15 to about 25, about 20 to about 40, about 20 to about 25 or about 30 to about 40 Nucleotide.
In certain embodiments, STAR primer is forward primer.In certain embodiments, STAR primer is reverse primer.In certain embodiments, STAR sequence label is completely contained in the second PBR.In certain embodiments, STAR sequence label is completely contained in (that is, in the region having target nucleic acid to be amplified) in the target sequence downstream of the second PBR.In certain embodiments, STAR sequence label bridge joint second PBR with have target nucleic acid to be amplified.In certain embodiments, STAR sequence label can be 0,1,2,3,4,5,6,7,8,9,10,11,12 or 13 Nucleotide with the overlapping of the second PBR.Preferably, this overlap is 11,12 or 13 Nucleotide.In a particular embodiment, STAR sequence label is 12 Nucleotide with the overlapping of the second PBR.In certain embodiments, STAR sequence label can be 0,1,2,3,4,5,6,7,8,9,10,11,12 or 13 Nucleotide with the overlapping of target sequence downstream of the second PBR.Preferably, this overlap is 11,12 or 13 Nucleotide.In a particular embodiment, STAR sequence label is 12 Nucleotide with the overlapping of target sequence downstream of the second PBR.In certain embodiments, STAR sequence label mates completely with target nucleic acid.In certain embodiments, STAR sequence label contains the mispairing with target nucleic acid.
In certain embodiments, STAR primer can be used as and strengthen the specific general instrument easily again of PCR.STAR primer can be widely used in gene type, single nucleotide polymorphism (SNP) detects, Rare allele detects, the pre-amplification of mutant sequence, rare mutation health check-up survey, DNA methylation analysis, unwanted background dna selectivity containment, library subtraction etc. in.In addition, STAR primer may be used for the connection adapter connected in the pre-amplification of the miRNA connected at adapter selective amplification containment and with miRNAs
analyze and distinguish (referring to the U.S. Provisional Patent Application owning CO-PENDING together the 61/721st that on November 2nd, 2012 submits to, No. 968, No. PCT/USxx/xxxxxxth, the PCT application that the name submitted to concurrently is therewith called " tiny RNA is caught, detect and quantitatively (Small RNA Capture; Detection and Quantification) ", each mode quoted in full in described application is incorporated herein.
In certain embodiments, be provided for the method for the improper amplification suppressing or reduce in fact target nucleic acid, described method comprises makes target nucleic acid contact with one or more STAR primer, and wherein said STAR primer comprises 5'STAR sequence label; And extend STAR primer to form extension products, extension products suppresses or substantially reduces the improper amplification of target nucleic acid whereby.
In certain embodiments, be provided for the method detecting one or more target nucleic acid molecules, described method comprises makes one or more target nucleic acid molecules and one or more STAR primer hybridization, and wherein said STAR primer comprises 5'STAR sequence label; Extend STAR primer to form one or more extension products; Amplification extension products is to form one or more amplified production; And detect at least one amplified production of presence or absence, detect one or more target nucleic acid molecules thus.
In certain embodiments, be provided for the method detecting one or more target nucleic acid molecules, described method comprises makes one or more target nucleic acid molecules and one or more STAR primer hybridization, and wherein said STAR primer comprises 5'STAR sequence label; Extend STAR primer to form one or more extension products; Increase extension products when Existing detector probe to form one or more amplified production, and wherein said detector probe comprises at least one Nucleotide of STAR primer; And detect at least one amplified production of presence or absence, detect one or more target nucleic acid molecules thus.
In certain embodiments, be provided for the method detecting two or more different target nucleic acid molecule from single hybridization, described method comprises makes a STAR primer and first object nucleic acid and the 2nd STAR primer and the hybridization of the second target nucleic acid, wherein a STAR primer hybridization to first object nucleic acid and the 2nd STAR primer hybridization to the second target nucleic acid, extend a STAR primer and the 2nd STAR primer to form extension products, extension products is assigned in the first amplified reaction to form the first amplified production and to assign in the second amplified reaction to form the second amplified production, primer wherein in the first amplified reaction corresponds to first object nucleic acid instead of the second target nucleic acid and primer in the second amplified reaction corresponds to the second target nucleic acid instead of the first nucleic acid, the first detector probe wherein in the first amplified reaction is different from the second detector probe in the second amplified reaction, wherein the first detector probe comprises at least one Nucleotide of a STAR primer and the second detector probe comprises at least one Nucleotide of the 2nd STAR primer, and detect two different target nucleic acid.
In certain embodiments, be provided for distinguishing two not homoallelic methods in target nucleic acid, described method comprise by described target nucleic acid and a STAR primer and the 2nd STAR primer and hybridize to described target nucleic acid common oppositely or forward primer combine, a wherein said STAR primer comprises corresponding to the first allelic STAR sequence label, and the 2nd STAR primer comprises corresponding to the second allelic STAR sequence label; Extend the first and second STAR primers respectively to form the first and second extension products; The first and second extension products increase respectively to form the first and second amplified productions; And detect presence or absence first and second amplified production, wherein the existence of the first amplified production represents the second allelic existence and the existence of the second amplified production represents the first allelic existence.For example, STAR design of primers may be used for general by what be incorporated in STAR primer
probe is to carrying out genotypic analyses (referring to Fig. 2 C).
For example, use reaction, with two STAR primers and hybridize to locus target nucleic acid common oppositely or forward primer carry out in amplification of nucleic acid sample specific gene seat, STAR primer containing the STAR sequence label with the first allelic complementation of described locus and another STAR primer containing the STAR sequence label with the second allelic complementation of described locus; Extend STAR primer to form extension products; Amplification extension products is to form amplified production; And detect presence or absence amplified production.The detector probe through marking with STAR primer hybridization, such as TaqMan probe can be used.In certain embodiments, detector probe is different from the first and second STAR primers.For example, use the detector probe through a kind of dye marker together with a STAR primer and use the detector probe marked through a kind of different dyes together with the 2nd STAR primer.
When there is the first allelotrope in nucleic acid samples, use and will produce the extension products of wherein STAR sequence label and the complementation of labeled targets district containing being designed to the STAR primer of the STAR sequence label of the first allelic sequences complementation, allow extension products inflection self-annealing to become stem-ring structure.Therefore, the amplification comprising the first allelic extension products is curbed and the amount of amplified production greatly reduces or there is not amplified production.The extension products of wherein STAR sequence label and the mispairing of labeled targets district will be produced with the STAR primer of the STAR sequence label of the second allelic sequences complementation containing being designed to.With this mispairing, comprise the second allelic extension products not self-annealing, or at least do not reach the degree being enough to contain amplification.Therefore, for not the existing and represent to there is the first allelotrope in target nucleic acid sample for the existence of the amplified production (" the second amplified allele product ") of the second allelic STAR primer of amplified production (" the first amplified allele product ") of the first allelic STAR primer.Existence and not the existing of the second amplified allele product of the first amplified allele product represent to there is the second allelotrope in target nucleic acid sample.
In certain embodiments, be provided for distinguishing the method methylated with non-methylated targets nucleic acid.Use the genomic dna of bisulfite conversion, STAR primer may be used for carrying out quantitatively DNA methylation.Referring to such as Fig. 2 B.STAR primer can be designed to contain methylate DNA amplification by the dC in target target nucleic acid, or for containing that non-methylate DNA increases by the dU in target target nucleic acid.
For example, use reaction, with STAR primer and hybridize to through bisulf iotate-treated target nucleic acid common oppositely or forward primer to increase described target nucleic acid, wherein said STAR primer comprises the STAR sequence label corresponding to the non-STAR labeled targets sequence that methylates in target nucleic acid; Extend STAR primer to form extension products; Amplification extension products is to form amplified production; And detect presence or absence amplified production, wherein the existence of amplified production represents methylated existence and not existing of amplified production represents methylated and do not exist.
Non-methylated cytosine is converted into uridylic and does not change methylated cytosine by bisulf iotate-treated.When target nucleic acid is when the nucleotide base place will inquiring its methylation state is non-methylating, use containing be designed to the STAR sequence label of non-methylated targets complementary (such as, comprise with in STAR labeled targets district at the STAR sequence label of " A " of the dU complementation through transforming at inquired nucleotide base place) STAR primer will produce the extension products of wherein STAR sequence label and the complementation of labeled targets district, allow extension products inflection self-annealing to become stem-ring structure.Therefore, the amplification of extension products is curbed and the amount of amplified production greatly reduces or there is not amplified production, illustrates thus not exist in target nucleic acid to methylate.When target nucleic acid to inquire the nucleotide base place of its methylation state be methylate time, use the extension products containing will produce wherein STAR sequence label and labeled targets district mispairing (" A " of STAR sequence label and " C " in STAR labeled targets district) for the STAR primer of the STAR sequence label of non-methylated targets sequence.With this mispairing, extension products not self-annealing, or at least do not reach the degree being enough to contain amplification.Therefore, the existence of amplified production represents methylating in target nucleic acid.
In another embodiment, use similar reaction, with the STAR primer amplification DNA comprising methylate DNA specificity STAR sequence label.When target dna is methylated, use containing be designed to the STAR sequence label of methylated targets complementary (such as, comprise with STAR labeled targets district in inquire the STAR sequence label of " G " of dC complementation at nucleotide base place of methylation status) STAR primer will produce the extension products of wherein STAR sequence label and the complementation of labeled targets district.This complementarity allows extension products inflection and self-annealing becomes stem-ring structure.Therefore, the amplification of extension products is curbed and the amount of amplified production greatly reduces or there is not amplified production, illustrates thus to exist in target nucleic acid to methylate.When target nucleic acid is when the nucleotide base place will inquiring its methylation state is non-methylating, use the extension products that will produce wherein STAR sequence label and labeled targets district mispairing (" G " of STAR sequence label and " U " in STAR labeled targets district) containing the STAR primer for the STAR sequence label of non-methylated targets sequence.With this mispairing, extension products not self-annealing, or at least do not reach the degree being enough to contain amplification.Therefore, to represent in target nucleic acid non-methylates in the existence of amplified production.
In another embodiment, combine two reactions to carry out quantitatively DNA methylation.
In certain embodiments, provide composition, wherein said composition comprises one or more nucleic acid molecule and at least one oligonucleotide, and wherein said oligonucleotide comprises STAR sequence label and wherein said oligonucleotide is oligonucleotide of the present invention.In certain embodiments, described composition comprises nucleic acid polymerase further.In certain embodiments, described composition comprises primer forward or backwards further.In certain embodiments, described composition comprises detector probe further.
In certain embodiments, provide test kit, described test kit may be used for using described oligonucleotide to carry out hybridizing, extending and amplified reaction herein.Preferred test kit can comprise one or more and is configured for the container (such as bottle, pipe etc.) that comprises for the reagent in described method herein and optionally can comprises the specification sheets or scheme that use described reagent.Test kit described herein can comprise one or more and be selected from by the component of the following group formed: one or more oligonucleotide described herein (includes, but is not limited to STAR primer, forward and reverse primer and detector probe), one or more archaeal dna polymerase (such as heat-stabilised poly synthase), one or more reversed transcriptive enzyme or other DNA any or RNA polymerase, one or more can one or more reagent in mark described in quencher, one or more damping fluid or buffering salt, one or more Nucleotide, (it may be used for assaying reaction performance to one or more target/template molecule, i.e. control reaction) and other reagent for analyzing or manipulate further product or the intermediate produced by described method herein.Described additional component can comprise component for cloning and/or check order and for detecting or component quantitatively needed for Related Nucleic Acid Molecules or equipment.
In certain embodiments, provide the test kit being applicable to synthetic nucleic acid molecule further, described test kit comprises one or more oligonucleotide disclosed herein, comprises STAR primer.In certain embodiments, provide the test kit being applicable to amplifier nucleic acid molecule, described test kit comprises one or more oligonucleotide disclosed herein, comprises STAR primer.In certain embodiments, be provided for the test kit detecting or measure nucleic acid synthesis or amplified production, described test kit comprises one or more oligonucleotide disclosed herein, comprises STAR primer.
In order to more clear and describe concisely and point out theme of the present invention, provide particular term used in the following description and the appended claims book to give a definition.Run through this specification sheets, illustrating of particular term be regarded as limiting examples.
Unless otherwise specified exactly, otherwise as this specification sheets use, word " (a/an) " means at least one.In this manual, unless otherwise specified exactly, otherwise the use of odd number comprises plural number.For example, but be not as restriction, " target nucleic acid " means there is more than one target nucleic acid; For example, one or more copy and two or more different target nucleic acid materials of a specific objective nucleic acid substances.The term "and/or" term meant before and after oblique line can connect together or independent.In order to illustrate, but be not as restriction, " X and/or Y " can mean " X " or " Y " or " X " and " Y ".
Should be appreciated that to there is " about " of hint before the temperature discussed in the present invention, concentration, time etc., small and immaterial deviation is originally taught in the scope of content in this article.In addition, " comprise (comprise/comprises/comprising) ", " containing (contain/contains/containing) " and the use of " comprising (include/includes/including) " do not intend to be restrictive.Should be understood that above general description and following detailed description are only exemplary and indicative and do not limit and teach content.
Unless pointed out specially in above specification sheets, otherwise describe in above specification sheets the embodiment of " comprising " various component also contain " by " described component " forms " or " substantially by " described component " forms "; Describe in the description " by " embodiment that " forms " of various component also contains " comprising " described component or " substantially by " described component " forms "; And describe in the description the embodiment that " substantially by " various component " forms " also contain " by " described component " forms " or " comprising " described component (this interchangeability is not suitable for the use in detail in the claims of these terms).
Chapter title used herein only for organizational goal and should not be construed as by any way restriction institute want theme.The all documents quoted in this specification sheets, include, but is not limited to patent, patent application, article, books and paper, and the mode all quoted in full with it for any object is incorporated herein clearly.In the conflicting situation of arbitrary term defined in any one and this specification sheets in be incorporated to document, be as the criterion with this specification sheets.Although describe in conjunction with each embodiment and originally teach content, not intending originally teaching content constraints is described embodiment.On the contrary, originally teach content as it be to be appreciated that those skilled in the art that and contain various replacement scheme, amendment and equivalent.
" amplicon " and " amplified production " generally refers to the product of amplified reaction as used herein, the term.Amplicon can be double-strand or strand, and can comprise the component chain separated by making the sex change of double-stranded amplification product obtain.In certain embodiments, the amplicon of an amplification cycles can serve as the template in a rear amplification cycles.
Term " annealing (annealing) " and " hybridization (hybridizing) " include, but is not limited to the variant of root " hybridization (hybridize) " and " annealing (anneal) ", use interchangeably and mean a nucleic acid and the nucleotide base of another nucleic acid and match mutually use mutually, described interaction impels formation double helix, triple helical body or other higher ordered structure.According to Wo Sen-Ke Like (Watson-Crick) and Hu Sitan (Hoogsteen) type hydrogen bond knot, elementary mutually mutually with usually having nucleotide base specificity, such as A:T, A:U and G:C.In certain embodiments, base stacking and hydrophobic interaction also can facilitate double helix stability.Primer and probe anneals are well-known to the condition of complementary sequence in the art, such as " practical approach of nucleic acid hybridization " (
nucleic Acid hybridization, A Practical Approach), breathe out Metz (Hames) and John Higgins (Higgins) volume, IRL press, Washington DC (Washington, D.C.) (1985) and Wei Temo (Wetmur) and Dai Weisen (Davidson), " molecular biology " (
mol.Biol.) described in 31:349 (1968).
In general, whether described annealing is carried out especially by the impact of following each: the length of the complementary portion of the complementary portion of primer and its corresponding binding site in target flanking sequence and/or amplicon, or the corresponding complementary part of reporter probe and the length of its binding site; PH; Temperature; Monovalence and divalent cation there is situation; The ratio of G and C Nucleotide in hybridization region; The viscosity of substratum; And there is situation in denaturing agent.Time needed for described variable impact hybridization.Therefore, preferred annealing conditions will depend on application-specific and determine.But when invariably when testing, described condition can be determined by those of ordinary skill in the art routinely.Preferably, select following annealing conditions: allow primer and/or probe optionally with the complementary sequence hybridization in corresponding target flanking sequence or amplicon, but under the second temperature of reaction, do not reach any significance degree with the different target nucleic acid in response composite or non-targeted sequence hybridization.
Term " is optionally hybridized " and its variant means under appropriate stringency, specified sequence (such as (but being not limited to) primer) and the second sequence (such as the primer binding site of (but being not limited to) target flanking sequence or amplicon) comprising a series of complementary nucleotide are annealed, but it is unannealed such as, to improper sequence, non-targeted nucleic acid, probe or other primer.Usually, along with the melting temperature(Tm) of temperature of reaction towards specific double-stranded sequence increases, the relative quantity of selective cross generally increases and mistake causes general minimizing.In this manual, the statement of a sequence and another sequence hybridization or selective cross contains that wherein two sequences are all overall hybridize or the situation of selective cross each other, and in wherein said sequence one or two only a part hybridize or selective cross to the situation of a part for another complete sequence or another sequence.
As used herein, term " severity " is for being defined in the solvent composition by the temperature that formed during the hybridization of the mixture comprising two complementary nucleotide sequences and subsequent process steps and existence.The stability of hybridization that severity also defines the amount of homology, requirement and formed between two nucleotide sequences.Along with stringency increases, selective cross is favourable and non-specific cross hybridization is disadvantageous.Relative under the lower stringency of initiation that more may make a mistake, the stringency of increase corresponds to higher cultivation temperature, lower salt concn and/or higher pH usually.Those skilled in the art understands, enable primer or primer pair selective cross to the appropriate stringency of corresponding target flanking sequence and/or amplicon can use well-known technology and determine routinely when invariably when experiment (referring to such as " PCR: the basis from backstage to worktable " (
pCR:The Basics from background to Bench), McPpherson (McPherson) and Moller (Moller), this Science Press of Baeyer (Bios Scientific Publishers), 2000).
" or its combination " refers in all arrangements of described term term listed hereinbefore and combination as used herein, the term.For example, " A, B, C or its combination " intend to comprise in the following at least one: A, B, C, AB, AC, BC or ABC, and if order is important under specific circumstances, so also have BA, CA, CB, ACB, CBA, BCA, BAC or CAB.Continue this example, comprise the combination of the repetition containing one or more project or term clearly, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB etc.Skilled people in the industry will understand, unless apparent from context in addition, otherwise usually there are not the several quantitative limitations to the project in any combination or term.
Be converted into any process of two single stranded polynucleotide or a strand or single stranded polynucleotide in fact when " sex change (denaturing/denaturation) " refers to that wherein double-stranded polynucleotide is suitable as used herein, the term, described double-stranded polynucleotide includes, but is not limited to comprise genomic dna (gDNA) fragment of at least one target nucleic acid, double stranded amplicon or comprises the polynucleotide of at least one double-strand section.Make double-stranded polynucleotide sex change include, but is not limited to make double-strandednucleic acid to become strand or the various thermal technology of strand and chemical technology in fact, such as (but being not limited to) discharges the independent single chain components of two of double-stranded polynucleotide or the double helix comprising two kinds of oligonucleotide.Those skilled in the art will appreciate that adopted sex change technology is not generally restrictive, unless it disturbs in fact subsequent anneal or the enzymatic step of amplified reaction, or in some method, disturb the detection of fluorescent signal.
As used herein, term " Tm " is used in reference to melting temperature(Tm).Melting temperature(Tm) is that a group double chain acid molecule becomes the temperature being partly dissociated into strand.
" minor groove binders " refers to and sometimes embeds the small molecules in the ditch of double-stranded DNA with sequence-specific fashion as used herein, the term.In general, minor groove binders can adopt crescent shape and therefore embed closely in double-helical ditch, the not only long but also flat molecule of usual transfer water.Minor groove binding molecule comprises several aromatic rings connected by reversing key freely usually, such as (but being not limited to) furans, benzene or pyrrole ring.
Term " terminal " is measured and is referred to that wherein data gathering occurs over just the method after reaction has stopped.
Term " in real time " and " real-time continuous " be interchangeable and refer to wherein data gathering occur in polymerization process by periodic monitoring during method.Therefore, amplification is become one step with combining data detection by described method.
As used herein, term " quantitative PCR " refers to that using PCR to carry out quantitate gene expresses.
As used herein, term " C
t" and " cycle threshold " refer to that fluorescence intensity is greater than the time of background fluorescence.It is characterized in that the time point (or PCR circulation) target amplification being detected for the first time.Therefore, in initial substance, the quantity of target dna is larger, and the remarkable increase of fluorescent signal will occur faster, produces lower C
t.
As used herein, term " primer " refers to synthesis mode or with the single stranded oligonucleotide of biological mode generation, is extended between its amplification at nucleic acid molecule or polymerization period by the covalently bonded of nucleotide monomer.Nucleic acid amplification is normally based on the nucleic acid synthesis undertaken by nucleic acid polymerase or reversed transcriptive enzyme.Many described polysaccharases or reversed transcriptive enzyme need to there is the primer that can synthesize to cause described nucleic acid through extension.Primer is 11 bases or longer normally; Most preferably, primer is 17 bases or longer, but depends on needs and determine, and can use shorter or longer primer.As those skilled in the art will appreciate that, oligonucleotide disclosed herein can be used as one or more primer in various extension, synthesis or amplified reaction.
Term " complementarity " and " complementation " are interchangeable and refer to that polynucleotide form the ability of base pair each other.Base pair is formed by the hydrogen bond between the nucleotide units in antiparallel polynucleotide chain or region usually.Complementary polynucleotide chain or region can by Wo Sen-Ke Like modes base pairing (such as, A and T, A and U, C and G).100% complementarity refers to the situation that each nucleotide units in one of them polynucleotide chain or region can be tied with each nucleotide units generation hydrogen bond in the second polynucleotide chain or region.The situation of hydrogen bond knot can be there is each other in some but the not all nucleotide units that " are less than complete complementary " and refer to wherein two chains or two unit.
As used herein, term " reverse complementary sequence " refers to according to the rule that defined by Watson-Crick base pairing and the double-helical antiparallel character of DNA-DNA, RNA-RNA and RNA-DNA, by annealing/base pairing or in fact annealing/base pairing to the sequence of the second oligonucleotide.Therefore, as an example, the reverse complementary sequence of RNA sequence 5'-AAUUUGC will be 5'-GCAAAUU.Substituting base pair scheme can also be comprised at reverse complementary sequence, include, but is not limited to G-U pairing.
As used herein, term " probe " refers to by designing or selecting, containing allow its under limited severity specifically (that is, preferentially) hybridize to the synthesis of the specific nucleotide sequences of target nucleic acid sequence or with biological mode produce nucleic acid (DNA or RNA).
As used herein, " more non-extensible in fact " for describing when the 3' least significant end Nucleotide of oligonucleotide is not complementary with the corresponding base of target/template nucleic acid, in the feature of oligonucleotide extended and/or extend inefficiently in amplified reaction or do not extend.
As used herein, term " template " can exchange with " target molecule " or " target nucleic acid " and refer to be amplified, copy or the double-strand that extends, synthesize or check order or single stranded nucleic acid molecule.When double chain DNA molecule, its chain sex change formation Article 1 and Article 2 chain is made to increase to these molecules, check order or synthesize.To hybridize under proper condition with the primer of the part complementation of template and then polysaccharase (archaeal dna polymerase or reversed transcriptive enzyme) can synthesize the nucleic acid molecule with described template or one partial complementarity.According to the present invention, the length of the molecule of new synthesis can be equal with original template or shorter than it.Mispairing between the synthesis or extended peroid of the molecule of new synthesis membership produce the base pair of one or more mispairing.Therefore, synthesis molecule need not with template complete complementary.Template can be RNA molecule, DNA molecular or DNA/RNA mixed molecules.The molecule of new synthesis can serve as the template of the synthesis of follow-up nucleic acid or amplification.
As used herein, term " extension " refers to that the primer extension wherein hybridizing to target nucleic acid reacts with the prolongation of " extension products " of the chain of target nucleic acid complementation to be formed to comprise.
Target nucleic acid can obtain from any source, and can comprise any amount of different composition component.For example, target can be nucleic acid (such as, DNA or RNA), transfer RNA (tRNA) (tRNA), siRNA (siRNA), Microrna (miRNA) or other ripe tiny RNA, and nucleic acid analog or other nucleic acid mimics can be comprised.Target can through methylating, non-ly to methylate or both.Target can be through bisulf iotate-treated and the non-methylated cytosine(Cyt) being converted into uridylic.In addition, should be appreciated that " target nucleic acid " can refer to target nucleic acid itself and its sub (such as amplified production) and native sequences.Originally the target molecule teaching content can derive from any amount of source, includes, but is not limited to virus, archeobacteria, protobiont, prokaryotic organism and eukaryote, such as (but being not limited to) plant, fungi and animal.These sources can include, but is not limited to whole blood, biopsy, lymph, marrow, amniotic fluid, hair, skin, seminal fluid, biological warfare agent, anus secretory product, vaginal secretions, sweat, saliva, buccal swab, various environmental sample (such as, agricultural, water and soil), general Study sample, general purified sample, through culturing cell and dissolved cell.Should be appreciated that, target nucleic acid can use in various program as known in the art any one be separated from sample, described program is applying biological system (Applied Biosystems) ABI such as
6100 nucleic acid preparation station (Life Technologies, Inc. (Life Technologies Corp., Carlsbad, CA) of Carlsbad, CA and ABI
6700 automatic nucleic acid workstations (Life Technologies, Inc.),
mirVana
tMrNA separating kit (Life Technologies, Inc.) etc.Should be appreciated that, target nucleic acid can carry out cutting or shearing before analysis, comprises and uses following program: such as mechanical force, sonication, restriction endonuclease cracking or any method as known in the art.In general, the target nucleic acid originally teaching content will be strand, but in certain embodiments, target nucleic acid can be double-strand, and strand can be produced by sex change.
As used herein, term " hair clip " and " stem-ring " are interchangeable and for representing that wherein one or more part of oligonucleotide and one or more other parts of oligonucleotide form the oligonucleotide structure of base pair.When two number of base pairs form the double stranded section of oligonucleotide, described double stranded section can be referred to as stem.Therefore, depend on the quantity of complementary portion used, multiple stem (preferably, about 1 to about 10) can be formed.
" be incorporated to " part meaning to become DNA or RNA molecule or primer as used herein, the term.
As used herein, the term " nucleic acid binding dye " refer to double-stranded polynucleotide have specificity or at least with double-stranded polynucleotide in conjunction with time demonstrate than with single stranded polynucleotide in conjunction with time larger in fact Fluorescence Increasing fluorescence molecule.Usually, nucleic acid binding dye molecule is combined with described double-strand section by between the base pair that is inserted into the double-strand section of polynucleotide, but be combined in the major groove of described double-strand section or ditch or both in the middle of.The limiting examples of nucleic acid binding dye comprises ethidium bromide; DAPI; Hirst derivative (Hoechst derivatives), includes, but is not limited to Hirst 33258 and Hirst 33342; Intercalating agent, (such as (but being not limited to) carries two fluorescence four tooth beta-diketon-Eu to comprise lanthanide chelate
3+inner complex (NDI-(BHHCT-Eu
3+)
2) benzene-naphthalene diimide derivative, referring to people such as such as wild island (Nojima), " nucleic acids research " (
nucl.Acids Res.) supplementary issue the 1st phase 105 (2001)) and some asymmetric cyanine dyes, such as
green and
As used herein, term " polynucleotide ", " oligonucleotide " and " nucleic acid " use interchangeably and refer to strand and the dichain polymer of nucleotide monomer, include, but is not limited to the 2'-deoxyribonucleotide (DNA) that connected by internucleotide phosphate diester linkage binding or internucleotide analogs and associated counter ions and ribonucleotide (RNA), described counter ion such as H
+, NH
4 +, trialkyl ammonium, Mg
2+, Na
+deng.Polynucleotide can be made up of deoxyribonucleotide completely, be completely made up of ribonucleotide or be made up of its chimeric mixtures, and can comprise nucleotide analog.Nucleotide monomer units can comprise any one in described Nucleotide herein, includes, but is not limited to Nucleotide and/or nucleotide analog.Usually from several monomeric unit to several thousand monomeric nucleotide units not etc., such as 5-40, when they are called as oligonucleotide sometimes in the art for the size of polynucleotide.Unless otherwise noted, otherwise whenever presenting polynucleotide sequence, all should be appreciated that, Nucleotide is from left to right order by 5' to 3' and unless otherwise noted, otherwise " A " represents Desoxyadenosine, " C " represents Deoxyribose cytidine, and " G " represents pancreatic desoxyribonuclease, " T " represents deoxythymidine, and " U " represents deoxyuridine.
Term " Nucleotide " refers to the phosphoric acid ester of nucleosides, such as triguaiacyl phosphate, and wherein modal esteratic site is the hydroxyl of the C-5 position being connected to pentose.
Term " nucleosides " refers to the compound be made up of the purine of the 1' position being connected to pentose (comprising 2'-deoxidation and 2'-OH-form), deazapurine or pyrimidine nucleoside base, and described base is VITAMIN B4, guanine, cytosine(Cyt), uridylic, thymus pyrimidine, denitrogenation VITAMIN B4, denitrogenation guanosine etc. such as.When nucleoside base be purine or 7-deazapurine time, pentose is connected to 9 core bases of purine or deazapurine, and when core base is pyrimidine, pentose is connected to 1 core base of pyrimidine.
Term " stand-in " comprises the synthetic analogues having modified base part, modify sugar moieties and/or modification phosphonate moiety.Phosphoric acid ester analogue generally comprises one or more in+5 oxidation state and in Sauerstoffatom of wherein phosphorus atom by the phosphoric acid ester analogue of the non-oxygen aliquot replacement of such as sulphur.Exemplary phosphoric acid ester analogue comprises: thiophosphatephosphorothioate, phosphorodithioate, phosphoroselenoate, two phosphoroselenoate, aniline thiophosphatephosphorothioate, aniline phosphoric acid ester, phosphoramidate, borono-phosphoric acid ester, comprise associated counter ions, such as H
+, NH
4 +, Na
+.Exemplary base analogue comprises: 2,6-diaminopurine, xanthoglobulin, pseudouridine, C-5-propine, iso-cytosine, isoguanine, 2-thiopyrimidine.Exemplary sugar analogue comprises: 2' modifies or 3' modifies, and wherein 2' position or 3' position are hydrogen, hydroxyl, alkoxyl group (such as methoxyl group, oxyethyl group, allyloxy, isopropoxy, butoxy, isobutoxy and phenoxy group), azido-, amino or alkylamino, fluorine, chlorine and bromine.
As used herein, term " reactor " generally refers to any container, chamber, device or the subassembly according to originally teaching content and can reacting wherein.In certain embodiments, reactor can be microtubule, such as (but being not limited to) 0.2mL or 0.5mL reaction tubes, such as
light pipe (Life Technologies, Inc. of Carlsbad, CA) or Eppendorf tube, or other container of that class in the convention of Molecular Biology Lab.In certain embodiments, reactor comprises the hole of porous plate, the passage of the point on slide glass or microfluidic device or chamber, includes, but is not limited to
low density array or
open array PCR in real time plate (all from Life Technologies, Inc.).For example, but not as restriction, multiple reactor can be there is on same upholder.In certain embodiments, such as (ion 316 can be comprised available from the chip lab shape device of Ka Lipo (Caliper), Fu Luda (Fluidigm) and Life Technologies, Inc.
tMwith ion 318
tMchip) reactor in disclosed method can be served as.To recognize, various reactor is the commercially available situation that maybe can be designed to be applicable to originally teach content.
Term " reporter group " uses in a broad sense in this article and refers to any identifiable label, mark or part.
Term " thermally-stabilised " refers to when mentioning that enzyme uses the enzyme because of heat inactivation with resistance (such as having the polypeptide of nucleic acid polymerization enzymic activity)." thermally-stabilised " enzyme and " thermally labile " polysaccharase are formed and contrast, and the latter can pass through thermal treatment inactivation.Thermally labile protein at inactivation under physiological temp, and can classify as intervening thermal stabilization (at about 45 DEG C to about 65 DEG C inactivation) and thermally-stabilised (being greater than inactivation at about 65 DEG C).For example, the activity of thermally labile T5 and T7DNA polysaccharase can by the constant temperature about 30 seconds complete deactivations described enzyme being exposed to about 90 DEG C.The resistance of heat-stabilised poly synthase activity to heat inactivation is greater than thermally labile polysaccharase.But heat-stabilised poly synthase does not intend to refer to the enzyme of complete heat resistanceheat resistant inactivation; Therefore, thermal treatment can make polymerase activity be reduced to a certain degree.The optimum temps of heat-stabilised poly synthase usually also will higher than thermally labile archaeal dna polymerase.
To perform the reagent concentration of specific function (amplification of such as nucleic acid molecule or digestion) under term " working concentration " refers to optimum concn used in the solution or close to described optimum concn.The working concentration of reagent is also described as " 1 × concentration " or " 1 × solution " (if reagent in the solution) of reagent equivalently.Therefore, the greater concn of reagent can also be described based on working concentration; For example, " 2 × concentration " or " 2 × solution " of reagent is defined as concentration up to the working concentration twice of reagent or solution; " 5 × concentration " or " 5 × solution " is up to working concentration five times etc.
As used herein, term " amplification (amplification) ", " nucleic acid amplification " or " amplification (amplifying) " refer to that the nucleotide sequence producing multiple nucleic acid-templated copy or produce multiple and nucleic acid-templated complementation copies.Described term (comprising term " polymerization ") can also refer to extend nucleic acid-templated (such as, by polymerization).Amplified reaction can be polymerase-mediated extension, such as polymerase chain reaction (PCR).But any known amplified reaction can be applicable to use as described herein.The term " amplification " that typically referring to target nucleic acid " index " increases may be used for both the linear of the selected target sequence quantity describing nucleic acid and index increase in this article.
Term " amplification reaction mixture " and/or " main mixture " can refer to the aqueous solution of various (some or all of) reagent comprised for the target nucleic acid that increases.Described reaction can also use solid support thing (such as, array) to perform.Described reaction can also need to perform with single or multiplex form according to user.These reactions generally include enzyme, aqueous buffer solution, salt, amplimer, target nucleic acid and ribonucleoside triphosphote.Depend on situation and determine, described mixture can be complete or incomplete amplification reaction mixture.Method for the target nucleic acid that increases can be the obtainable any method of those skilled in the art.Any external means that the copy of nucleic acid target sequence is doubled can be adopted.These means comprise linearly, logarithm and/or other amplification method any.Although the present invention can discuss PCR substantially as nucleic acid amplification reaction, but estimate that modification sanitising agent described herein should be effective in the nucleic acid amplification reaction of other type, the nucleic acid amplification reaction of other type described comprises polymerase-mediated amplified reaction (such as helicase dependent amplification (HDA), recombinase-polymeric enzymatic amplification (RPA) and rolling circle amplification (RCA)) and be connected amplified reaction (the such as Ligase detection reaction (LDR) mediated, ligase chain reaction (LCR) (LCR) and respective gap version) both, and the combination of nucleic acid amplification reaction, such as LDR and PCR is (referring to such as United States Patent (USP) 6, 797, 470).For example, modify sanitising agent and may be used for such as various connection in the reaction of mediation, in described reaction, adopt such as linking probe, contrary with PCR primer.Extra exemplary methods especially comprises polymerase chain reaction (PCR; Referring to such as No. the 4th, 683,202, United States Patent (USP); 4th, 683, No. 195; 4th, 965, No. 188; And/or the 5th, 035, No. 996), isothermal program (using the partial destruction (referring to such as No. 2006/087574, PCT publication WO) of one or more RNA polymerase (referring to such as No. 2006/081222, PCT publication WO), strand displacement (referring to such as No. RE39007Eth, United States Patent (USP)), primer molecule), ligase chain reaction (LCR) (LCR) (referring to people such as such as Wus (Wu), " genomics " (
genomics) 4:560-569 (1990)), and/or people's " institute of NAS periodical " such as Ba Lani (Barany) (
proc.Natl.Acad. sci.USA) 88:189-193 (1991)), Q β rna replicon enzyme system (referring to such as No. WO1994/016108th, PCT publication), based on rna transcription system (such as, TAS, 3SR), rolling circle amplification (RCA) is (referring to such as United States Patent (USP) the 5th, 854, No. 033; No. 2004/265897th, U.S. Patent Application Publication case; People's " natural genetics " such as Li Chadi (Lizardi) (
nat.Genet.) 19:225-232 (1998); And/or people's " nucleic acids research " such as Ba Na (Ban é r) (
nucleic Acid Res.), 26:5073-5078 (1998)) and strand displacement amplification (SDA) (people's " clinical chemistry " such as Arthur D. Little (Little) (
clin.Chem.) 45:777-784 (1999)).These systems go for being polymerized and/or increasing the target nucleic acid used as described herein together with obtainable other systems many of skilled people in the industry.
" amplification efficiency " can refer to can through quantitative with the spawn measuring copy number (such as, described term can refer to pcr amplification, LCR connection product and/or similar product).Amplification and/or polymerization efficiency can be measured by various method as known in the art, described method includes, but is not limited to measure calibration dilution curve and slope calculating, use people such as graceful in haler this (Hellemans), " genome biology " (
genome Biology) the qBase software mensuration described in 8:R19 (2007), use as Li Wake (Livak) and Shi meter Te Gen (Schmittgen), " method " (
methods) the Δ Δ Cq computing method mensuration described in 25:402 (2001), or pass through as Pfaff (Pfaffl), " nucleic acids research " (
nucl. acids Res.) method described in 29:e45 (2001), all these mode quoted in full with it is incorporated herein.
In certain embodiments, amplification technique comprises at least one amplification cycles, such as (but being not limited to) following steps: make double-strandednucleic acid sex change with separated portion chain; Primer hybridization is made to arrive the primer binding site (or any one complementary sequence) time suitably of target flanking sequence or amplicon; And use archaeal dna polymerase with template dependent manner synthesizing ribonucleotide chain.Described circulation can or can not repeat.In certain embodiments, amplification cycles comprises multiple amplification cycles, such as (but being not limited to) 20 amplification cycles, 25 amplification cycles, 30 amplification cycles, 35 amplification cycles, 40 amplification cycles, 45 amplification cycles or be greater than 45 amplification cycles.
In certain embodiments, amplification comprises use instrument and carries out thermal cycling, and described instrument is (but being not limited to) such as
pCR system 9700,9600,2700 or 2400 thermal cycler; Applied
viiA
tM7 real-time PCR systems; Applied
7500 Fast real-time PCR system; 7900HT Fast real-time PCR system etc. (all can purchased from Life Technologies, Inc. of Carlsbad, CA).In certain embodiments, in amplified reaction, produce single-stranded amplicon, described amplified reaction is (but being not limited to) asymmetric PCR or A-PCR such as.
In certain embodiments, amplification comprises two-step reaction, include, but is not limited to pre-amplification step, in pre-amplification step, there is the amplification cycles (such as (but being not limited to) 2,3,4 or 5 amplification cycles) of limited quantity, then gained amplicon generally diluted and make diluted amplicon part in a rear amplification step, experience extra amplification cycles (referring to such as United States Patent (USP) the 6th, 605, No. 451 and No. 2004/0175733rd, U.S. Patent Application Publication case).
In certain embodiments, amplified reaction comprises multiplex amplification, wherein uses multiple different primers group synchronous amplification multiple different target nucleic acid and/or multiple different amplified production material.In certain embodiments, executed in parallel multiplex amplification reaction and single amplification reaction, comprise multiple substance or lower tuple reaction (such as (but being not limited to) is double, triple, quadruple, five weighs or sixfold reacts).
For to be polymerized and/or the exemplary methods of amplification of nucleic acid comprises such as polymerase-mediated extension.For example, polymerase-mediated extension can be polymerase chain reaction (PCR).In other embodiments, nucleic acid amplification reaction is multiple reaction.For example, be applicable to as described herein for being polymerized and/or increasing and to detect the exemplary methods of nucleic acid passable
form is purchased (referring to such as No. the 4th, 889,818, United States Patent (USP); 5th, 079, No. 352; 5th, 210, No. 015; 5th, 436, No. 134; 5th, 487, No. 972; 5th, 658, No. 751; 5th, 210, No. 015; 5th, 487, No. 972; 5th, 538, No. 848; 5th, 618, No. 711; 5th, 677, No. 152; 5th, 723, No. 591; 5th, 773, No. 258; 5th, 789, No. 224; 5th, 801, No. 155; 5th, 804, No. 375; 5th, 876, No. 930; 5th, 994, No. 056; 6th, 030, No. 787; 6th, 084, No. 102; 6th, 127, No. 155; 6th, 171, No. 785; 6th, 214, No. 979; 6th, 258, No. 569; 6th, 814, No. 934; 6th, 821, No. 727; 7th, 141, No. 377; And/or the 7th, 445, No. 900, all these mode hereby quoted in full with it is incorporated herein).
analyze usually by using the nucleic acid polymerase with 5' to 3' nuclease, the primer that can hybridize to herbicide-tolerant polynucleotide and relative to the oligonucleotide probe of described primer hybridization to described herbicide-tolerant polynucleotide 3', nucleic acid amplification can being performed to described herbicide-tolerant polynucleotide and carry out.Oligonucleotide probe generally includes detectable label (such as, fluorescent reporter molecule) and can the Quenching of fluorescence agent molecule of reporter molecules described in quencher.Usually, detectable label and quencher molecule are parts for Single probe.Along with amplification is carried out, polymerase digests probe is to be separated detectable label and quencher molecule.During reaction monitor detectable label (such as, fluorescence), the detection wherein marked corresponds to the generation (such as, signal is higher, and amplification amount is larger) of nucleic acid amplification.
variant (such as, the LNA analyzed
tMadditional
analyze) be well known in the art and will be applicable in method described herein.
As used herein, term " detector probe " refers to in amplified reaction, is generally used for quantitatively or the molecule of real-time PCR analysis and end point analysis.Described detector probe may be used for the amplification of monitoring objective polynucleotide.In certain embodiments, existing in amplified reaction detector probe is applicable to the amount of monitoring the amplicon produced in time.It is (described herein that described detector probe includes, but is not limited to 5'-Exonuclease analysis
probe is (also referring to United States Patent (USP) the 5th, 538, No. 848), various stem-toroidal molecule beacon is (referring to such as United States Patent (USP) the 6th, 103, No. 476 and the 5th, 925, No. 517 and sub-lucky (Tyagi) and carat silent (Kramer), " Nature Biotechnol " (
nature biotechnology) 14:303-308 (1996)), acaulescence or Linear Beacon (referring to such as No. 99/21881, PCT publication WO), PNA Molecular Beacons
tM(referring to such as No. the 6th, 355,421, United States Patent (USP) and the 6th, 593, No. 091), linear PNA beacon (referring to people such as such as Ku Bisi towers (Kubista),
sPIE4264:53-58 (2001)), non-FRET probe (referring to such as No. the 6th, 150,097, United States Patent (USP)),
probe (United States Patent (USP) the 6th, 548, No. 250), stem-ring and double helix Scorpion
tMprobe (people such as Suo Linasi (Solinas), " nucleic acids research " (
nucleic Acids Research) 29:E96 (2001) and United States Patent (USP) the 6th, 589, No. 743), bulge loop probe (United States Patent (USP) the 6th, 590, No. 091), false knot probe (United States Patent (USP) the 6th, 589, No. 250), cyclicons (United States Patent (USP) the 6th, 383, No. 752), MGB Eclipse
tMprobe (liking Bock bio-science (Epoch Biosciences)), hairpin probe (United States Patent (USP) the 6th, 596, No. 490), peptide nucleic acid(PNA) (PNA) luminous (light-up) probe, self-assembled nanometer particle probe and ferrocene modify probe, such as described in following each: United States Patent (USP) the 6th, 485, No. 901; Ma Halang adds people such as (Mhlanga), " method " (
methods) 25:463-471 (2001); The people such as Whitcomb (Whitcombe), " Nature Biotechnol " (
nature Biotechnology), 17:804-807 (1999); The people such as Isaksson (Isacsson), " molecular cell probe " (
molecular Cell Probes), 14:321-328 (2000); The people such as Si Wanweike (Svanvik), " analytical biochemistry " (
anal Biochem.) 281:26-35 (2000); The people such as Wolf (Wolffs), " biotechnology " (
biotechniques) 766:769-771 (2001); The people such as special soukous (Tsourkas), " nucleic acids research " (
nucleic Acids Research), 30:4208-4215 (2002); The people such as Li Qieli (Riccelli), " nucleic acids research " (
nucleic Acids Research) 30:4088-4093 (2002); Open people such as (Zhang), " Shanghai " (
shanghai.) 34:329-332 (2002); The people such as Max Wei Er (Maxwell), " U.S. chemical institute magazine " (
j.Am.Chem. soc.) 124:9606-9612 (2002); The people such as Blaw moral (Broude), " biotechnology trend " (
trends Biotechnol.) 20:249-56 (2002); People such as yellow (Huang), " chemical toxicology research " (
chem Res.Toxicol.) 15:118-126 (2002); And the people such as remaining (Yu), " American Chemical Society's will " (
j.Am.Chem.Soc) 14:11155-11161 (2001).Detector probe can also comprise quencher, includes, but is not limited to Black Hole Quencher (biological paddy hunter (Biosearch)), Iowa Black (IDT), QSY quencher (molecular phycobiliprotein complexes (Molecular Probes)) and dimethyl amino-azo-benzene formyl (Dabsyl) and Dabcel sulphonate/manthanoate quencher (love Bock).Detector probe can also comprise two probes, wherein such as fluorescent agent is on a probe, and quencher is on another probe, wherein two probes hybridize quencher signal together in target, or wherein hybridize in target by change fluorescence change signal characteristic.Detector probe can also comprise fluorescein(e) dye and SO
3instead of the sulfonate derivatives of carboxylate group, the phosphoramidite form of fluorescein, Cy5 phosphoramidite form (can purchased from such as General Electric's Medical Group (GE Healthcare)).In certain embodiments, use insert mark, such as ethidium bromide,
green I (Life Technologies, Inc. of Carlsbad, CA) and
(Life Technologies, Inc.), allows the real-time visual or visual at the terminal of amplified production when not Existing detector probe thus.In certain embodiments, real-time visual can comprise insertion detector probe and can adopt the detector probe based on sequence.In certain embodiments, by quencher at least in part when detector probe does not hybridize to complementary sequence in amplified reaction, and by non-quencher at least in part when hybridizing to complementary sequence in amplified reaction.In certain embodiments, the Tm originally teaching the detector probe of content is 63-69 DEG C, but should be appreciated that, by originally teaching the guiding of content, normal experiment can produce the detector probe with other Tm.In certain embodiments, probe can comprise various amendment further, such as minor groove binders (referring to such as United States Patent (USP) 6,486,308), for providing wanted thermodynamic characteristics further.In certain embodiments, detector probe can correspond to and differentiates part or differentiate partial complementarity sequence.
Another illustrative system being applicable to use as described herein utilize in substitution crossing method double-chain probe (referring to people's " analytical biochemistry " such as such as Morrison (Morrison) (
anal.Biochem.), 18:231-244 (1989); And/or people's " nucleic acids research " such as Lee (Li) (
nucleic Acids Res.), 30 (2, e5) (2002)).In the process, probe generally includes the complementary oligonucleotide that two have different lengths, and one of them comprises detectable label and another comprises quencher molecule.When not being attached to target nucleic acid, the signal of quencher containment detectable label.With target nucleic acid substitution crossing after, probe becomes detectable.Multiple probe can be used, each containing different detectable labels, to make it possible to inquire about multiple target nucleic acid in single reaction.
Be applicable to as described herein for being polymerized and/or increasing and the extra exemplary methods detecting target nucleic acid relates to " molecular beacon ", it is single-stranded hair-pin shape oligonucleotide probe.When there is target sequence, probe launch, combine and transmit (such as, fluorescing).Molecular beacon generally includes at least four kinds of components: 1) " ring ", the region of 18-30 Nucleotide, and it and target sequence are complementary; 2) two 5-7 Nucleotide " stem " is complimentary to one another in its either end being present in ring; 3) at 5' end, detectable label; And 4) at 3' end, quencher moieties, it (such as, is not attached to target nucleic acid) when probe is closed loop shape and prevents detectable label from transmitting.Therefore, when there is complementary target, " stem " part of beacon is separated, and impels probe hybridization to arrive target.Also the molecular beacon of other type known and they go in method described herein.Molecular beacon may be used in various analytical system.Described system is the amplification based on nucleotide sequence
for when without when temperature cycle by RNA polymerization and/or amplification to the single step isothermal process of double-stranded DNA.NASBA reaction needs avian myeloblastosis virus (AMV), reversed transcriptive enzyme (RT), t7 rna polymerase, RNA enzyme H and two Oligonucleolide primers usually.After amplification, molecular beacons detection can be used through the target nucleic acid of amplification.Other purposes of molecular beacon is well known in the art and will be applicable in method described herein.
Scorpions
tMsystem may be used for the exemplary analytical form of another kind in described method herein.Scorpions
tMprimer is bifunctional molecule, and wherein the undetectable quencher moieties of the fluorescence of primer and detectable label (such as, fluorophore) and quencher detectable label is covalently bound to probe together.When there is target nucleic acid, detectable label is separated with quencher, and this causes the signal launched from detectable label to increase.Usually, primer used in amplified reaction be included in the probe member of 5' end and " PCR blocker " element when hairpin loop starts (such as, six ethylene glycol (HEG) monomer (people's " Nature Biotechnol " such as Whitcomb (Whitcombe) (
nat.Biotech.) 17:804-807 (1999)).Probe is usually included in an end containing detectable label and in the oneself complementary stem sequence of another end containing quencher.In initial amplification cycles (such as, PCR), primer hybridization is to target and extend due to the effect of polysaccharase.Scorpions
tMsystem can use multiple can differently mark with the probe distinguished between probe, for checking and differential point sudden change.Use PCR as an example, after completing one and extending circulation, the target area of new synthesis will be connected to the chain identical with probe.After second sex change and anneal cycles, probe and target hybridization.Then hairpin hybridizes to a part for the new PCR primer produced.This causes detectable label to be separated with quencher and causes signal to launch.Described other purposes through label probe is well known in the art and will be applicable in method described herein.
In certain embodiments, described method performs before sequencing reaction or together with sequencing reaction.Term " order-checking " uses in a broad sense in this article and refers to that any technology of the order of at least some continuous nucleotide in polynucleotide (such as (but being not limited to) target nucleic acid or amplicon) is at least partially differentiated in known permission in this area.Some limiting examples of sequencing technologies comprise the dideoxy terminator method of Sang Ge (Sanger) and mark fills in nurse (Maxam) and the chemical cleavage method of gilbert (Gilbert), comprise the variant of those methods; Pass through sequencing by hybridization; Synthesis order-checking; And restriction enzyme digestion mapping.Some sequence measurements comprise electrophoresis, comprise capillary electrophoresis and gel electrophoresis; By sequencing by hybridization, comprise microarray hybridization; Mass spectrum; Single Molecule Detection; And ion/proton detects.In certain embodiments, order-checking comprises direct Sequencing, dual order-checking, cycle sequencing, single-basic extension order-checking (SBE), solid phase sequencing or its combination.In certain embodiments, order-checking comprises use instrument and detects order-checking product, and described instrument is (but being not limited to) ABI such as
377DNA sequenator; ABI
310,3100,3100-Avant, 3730 or 3730xl genetic analyzer; ABI
3700DNA analyser; Ion PGM
tMsequenator or Ion Proton
tMsequenator (all can purchased from Life Technologies, Inc. of Carlsbad, CA) or mass spectrograph.In certain embodiments, check order to comprise is incorporated to dNTP in amplified production, comprises dATP, dCTP, dGTP, dTTP, dUTP, dITP or its combination, and comprises the bi-deoxyribose nucleotide analog of dNTP.
Term " archaeal dna polymerase " is used in a broad sense in this article and refers to any polypeptide that can be extended with 5' to the 3' of template dependent manner catalysis hybridized primer by interpolation deoxyribonucleotide and/or some nucleotide analog.Such as (but being not limited to) adds deoxyribonucleotide successively to the 3' end being annealed to nucleic acid-templated primer during primer extension reaction.The limiting examples of archaeal dna polymerase comprises the archaeal dna polymerase of dependenc RNA, includes, but is not limited to reversed transcriptive enzyme; With the archaeal dna polymerase relying on DNA.Should be appreciated that, it is active and when amplified reaction comprises aggressive scission reaction that some archaeal dna polymerase (such as (but being not limited to) some eubacterium Aform DNA polysaccharase and Taq archaeal dna polymerase) can comprise structure specific nuclease further.
The nucleic acid polymerase that may be used in disclosed nucleic acid amplification reaction can be any nucleic acid polymerase carrying out reacting that works, and comprises such as procaryotic, fungi, virus, phage, plant and/or Eukaryotic nucleic acid polymerase.As used herein, term " archaeal dna polymerase " refers to the enzyme using nucleic acid chains as template de novo synthesis DNA chain.Archaeal dna polymerase uses existing DNA or RNA to synthesize template as DNA and along the polymerization of its template strand catalytic deoxidation ribonucleotide read.The DNA chain of new synthesis and template strand complementation.Archaeal dna polymerase only can add free nucleotide to the new 3'-hydroxyl terminal forming chain.It is by carrying out synthetic oligonucleotide by the 3'-hydroxyl that Nucleotide monophosphates transfers to from deoxynucleoside triphosphate (dNTP) oligonucleotide chain grown.This impels new chain to extend along the direction of 5' to 3'.Because archaeal dna polymerase only can add Nucleotide on the 3'-OH group be pre-existing in, so in order to start synthesis reaction of DNA, archaeal dna polymerase needs the primer that can add the first Nucleotide to it.Suitable primer can comprise oligonucleotide or its mosaic (such as, RNA/DNA chimeric primers) of RNA or DNA.Archaeal dna polymerase can be the varient of naturally occurring archaeal dna polymerase or the natural enzyme with above-mentioned activity.For example, it can comprise the archaeal dna polymerase with strand-displacement activity, the archaeal dna polymerase lacking 5' to 3' exonuclease activity, has the archaeal dna polymerase of reverse transcriptase activity or have the archaeal dna polymerase of endonuclease activity.
Any modified polysaccharase that suitable nucleic acid polymerase can also comprise holoenzyme, the Functional portions of holoenzyme, chimeric polysaccharase maybe can realize the synthesis of nucleic acid molecule.In the present invention, archaeal dna polymerase can also comprise polysaccharase, terminal enzyme (DNA), reversed transcriptive enzyme, Telomerase and/or Polyribonucleotide phosphorylase.The limiting examples of polysaccharase can comprise T7DNA polysaccharase, Eukaryotic mitochondria DNA polymerase γ, procaryotic DNA polymerase i, II, III, IV and/or V; Eukaryote Polymerase α, β, γ, δ, ε, η, ζ, ι and/or κ; Intestinal bacteria (E.coli) DNA polymerase i; E. coli dna polymerase III α and/or ε subunit; Escherichia coli polymerase IV, Escherichia coli polymerase V; Thermus aquaticus (T.aquaticus) DNA polymerase i; Bacstearothermophilus (B.stearothermophilus) DNA polymerase i; Wide ancient bacterium (Euryarchaeota) polysaccharase; Terminal deoxynucleotidyl transferase (TdT); Yeast saccharomyces cerevisiae (S.cerevisiae) polysaccharase 4; Across damage synthesized polymer enzyme; Reversed transcriptive enzyme; And/or Telomerase.The limiting examples of operable suitable heat-stable DNA polymerase includes, but is not limited to thermus thermophilus (Thermus thermophilus; Tth) archaeal dna polymerase, thermus aquaticus (Thermus aquaticus; Taq) archaeal dna polymerase, new Apollo are dwelt thermobacillus (Thermotoga neopolitana; Tne) archaeal dna polymerase, Thermotoga maritima (Thermotoga maritima; Tma) archaeal dna polymerase, beach is thermophilic coccus (Thermococcus litoralis; Tli or VENT
tM) archaeal dna polymerase, fierce fireball bacterium (Pyrococcus furiosus; Pfu) archaeal dna polymerase, DEEPVENT
tMarchaeal dna polymerase, Butterworth fireball bacterium (Pyrococcus woosii; Pwo) archaeal dna polymerase, bacstearothermophilus (Bacillus sterothermophilus; Bst) archaeal dna polymerase, extreme genus bacillus (Bacillus caldophilus; Bca) archaeal dna polymerase, sulfolobus acidocaldarius (Sulfobus acidocaldarius; Sac) archaeal dna polymerase, thermoplasma acidophilum (Thermoplasma acidophilum; Tac) archaeal dna polymerase, Huang are dwelt hot bacterium (Thermus flavus; Tfl/Tub) archaeal dna polymerase, redness are dwelt hot bacterium (Thermus ruber; Tru) archaeal dna polymerase, Bu Shi are dwelt hot bacterium (Thermus brockianus; DYNAZYME
tM) archaeal dna polymerase, addicted to hot autotrophic methane bacteria (Methanobacterium thermoautotrophicum; Mth) archaeal dna polymerase, mycobacterium (mycobacterium) archaeal dna polymerase (Mtb, Mlep) and its mutant and varient and derivative.RNA polymerase can also be used, such as T3, T5 and SP6 and its mutant, varient and derivative according to originally teaching content.In general, any I type archaeal dna polymerase can be used according to originally teaching content, but other archaeal dna polymerase can be used, include, but is not limited to the archaeal dna polymerase such as type III or family A, B, C.In addition, according to originally teach content can use any through genetic engineering modified archaeal dna polymerase, there is reduction or any archaeal dna polymerase (such as, SuperScript of inapparent 3' to 5' exonuclease activity
tMarchaeal dna polymerase) and/or through genetic engineering modified archaeal dna polymerase (such as, have the equivalent (such as, in Tth) of avtive spot sudden change F667Y or F667Y those,
fS, ThermoSequenase
tM),
golden,
taq archaeal dna polymerase, Therminator I, Therminator II, Therminator III, Therminator γ (all can purchased from New England's biology laboratory of Massachusetts Bei Fuli (New England Biolabs, Beverly, MA)) and/or its any derivative and fragment.As those skilled in the art will appreciate that, other nucleic acid polymerase also can be suitable.
Can be can usually along the direction of 5' to 3' from any enzyme of nucleic acid-templated synthetic nucleic acid molecule according to originally teaching the polysaccharase that content uses.Nucleic acid polymerase used in method disclosed herein can addicted to warm nature or thermophilic.Exemplaryly comprise T7DNA polysaccharase, T5DNA polysaccharase, Ke Lienuo (Klenow) fragment DNA polymerase, DNA polymerase i II etc. addicted to warm nature archaeal dna polymerase.May be used for the exemplary heat-stable DNA polymerase originally taught in the method for content and comprise Taq, Tne, Tma, Pfu, Tfl, Tth, Si Tuofeier fragment (Stoffel fragment), VENT
tMand DEEPVENT
tMarchaeal dna polymerase and its mutant, varient and derivative (United States Patent (USP) the 5th, 436, No. 149; United States Patent (USP) the 4th, 889, No. 818; United States Patent (USP) the 4th, 965, No. 188; United States Patent (USP) the 5th, 079, No. 352; United States Patent (USP) the 5th, 614, No. 365; United States Patent (USP) the 5th, 374, No. 553; United States Patent (USP) the 5th, 270, No. 179; United States Patent (USP) the 5th, 047, No. 342; United States Patent (USP) the 5th, 512, No. 462; No. 92/06188, PCT patent WO, No. 92/06200, WO and No. 96/10640, WO; Ba Erneisi (Barnes), " gene " (
gene) 112:29-35 (1992); The people such as Lao Ye (Lawyer), " PCR method and application " (
pCR Meth.Appl.) 2:275-287 (1993); The not people such as Raman (Flaman), " nucleic acids research " (
nucl.Acids Res.) 22 (15): 3259-3260 (1994)).The example lacking in fact the archaeal dna polymerase of 3' exonuclease activity includes, but is not limited to Taq, Tne (exo-), Tma (exo-), Pfu (exo-), Pwo (exo-) and Tth archaeal dna polymerase and its mutant, varient and derivative.
The archaeal dna polymerase be applicable in method disclosed herein can such as from Life Technologies, Inc.'s (Carlsbad, CA), Pharmacia (Pharmacia) (New Jersey Piscataway (Piscataway, N.J.)), Sigma (Sigma) (St. Louis (St.Louis, Mo.)) and Bao Lingman (Boehringer Mannheim) buy.
The enzyme be applicable in composition, method, composition and the test kit provided herein can also comprise any enzyme with reverse transcriptase activity.Described enzyme include, but is not limited to retroviral RTs, retrotransposon reversed transcriptive enzyme, viral hepatitis type b reversed transcriptive enzyme, cauliflower mosaic virus reversed transcriptive enzyme, bacterium reversed transcriptive enzyme, Tth archaeal dna polymerase, Taq archaeal dna polymerase (people such as Saiki (Saiki), " science " (
science) 239:487-491 (1988); United States Patent (USP) the 4th, 889, No. 818 and the 4th, 965, No. 188), Tne archaeal dna polymerase (No. 96/10640, PCT publication WO), Tma archaeal dna polymerase (United States Patent (USP) the 5th, 374, No. 553) and its mutant, fragment, varient or derivative (referring to such as all on September 9th, 1996 submit to own together, the U.S. Patent application the 08/706th of CO-PENDING, No. 702 and the 08/706th, No. 706, its mode quoted in full with it is incorporated herein).As one of ordinary skill in the art will appreciate that, modified reversed transcriptive enzyme can be obtained by restructuring well known in the art or genetic engineering technique with the archaeal dna polymerase with reverse transcriptase activity.Mutant reversed transcriptive enzyme or polysaccharase can such as make the transgenation of coding associated reverse transcription enzyme or polysaccharase to obtain by fixed point or random mutagenesis.Described sudden change can comprise point mutation, deletion mutantion and insertion mutation.In certain embodiments, use one or more point mutation (such as, replacing one or more amino acid with one or more different aminoacids) to construct and be applicable to mutant reversed transcriptive enzyme of the present invention or polysaccharase.The fragment of reversed transcriptive enzyme or polysaccharase by recombinant technology well known in the art by deletion mutantion, or can also use any one in multiple well-known proteolytic ferment, is obtained by the enzymic digestion of associated reverse transcription enzyme or polysaccharase.
In certain embodiments, the enzyme be applicable in the method that provides herein comprise that RNA enzyme H activity reduces or reduce in fact those.The described enzyme that RNA enzyme H activity reduces or reduces in fact by such as one or more point mutation as above, one or more deletion mutantion or one or more insertion mutation, can make the RNA enzyme H structure territory in associated reverse transcription enzyme suddenly change and obtains.The enzyme of " RNA enzyme H activity reduces in fact " refer to have be less than about 30%, be less than about 25%, be less than about 20%, be less than about 15%, be less than about 10%, be less than about 7.5% or be less than about 5% be less than about 5% or be less than about 2% corresponding wild-type or RNA enzyme H
+the enzyme of the RNA enzyme H activity of enzyme, described wild-type or RNA enzyme H
+enzyme is wild-type Moloney murine leukemia virus (Moloney Murine Leukemia Virus such as; M-MLV), avian myeloblastosis virus (AMV) or Rous sarcoma virus (Rous Sarcoma Virus; RSV) reversed transcriptive enzyme.The RNA enzyme H activity of any enzyme can be measured by various analysis, and described analysis is such as in such as No. the 5th, 244,797, United States Patent (USP); People such as Grzegorz Kotowiczs (Kotewicz), " nucleic acids research " (
nucl.Acids Res.) in 16:265 (1988); People such as Gerards (Gerard), " focusing " (
fOCUS) in 14 (5): 91 (1992); And at United States Patent (USP) the 5th, have described those in 668, No. 005, the disclosure of all these publications is incorporated herein by reference completely.
The polypeptide with reverse transcriptase activity be applicable in the method provided herein can such as be buied from Life Technologies, Inc.'s (Carlsbad, CA), Pharmacia (New Jersey Piscataway), Sigma's (St. Louis) or Bao Lingman biochemicals (Boehringer Mannheim Biochemicals) (Indianapolis, the state of Indiana (Indianapolis, Ind.)).Or, have reverse transcriptase activity polypeptide can according to those of ordinary skill in the art well-known for separating of being separated from its natural viral or bacterial origin with the standard program of purified native protein matter (referring to the such as people such as thatch (Houts) suddenly, " Journal of Virology " (
j.Virol.) 29:517 (1979)).In addition, have reverse transcriptase activity polypeptide can by those of ordinary skill in the art the recombinant DNA technology be familiar with prepare (referring to people such as such as Grzegorz Kotowiczs (Kotewicz), " nucleic acids research " (
nucl.Acids Res.) 16:265 (1988); The Sol base of a fruit this (Soltis) and Si Kaerka (Skalka), " institute of NAS periodical " (
proc.Natl. acad.Sci.USA) 85:3372-3376 (1988)).
The exemplified polypeptides with reverse transcriptase activity be applicable in the method provided herein comprises M-MLV reversed transcriptive enzyme, RSV reversed transcriptive enzyme, AMV reversed transcriptive enzyme, RAV (Rous Associated Virus; RAV) reversed transcriptive enzyme, myeloblastoma correlated virus (Myeloblastosis Associated Virus; MAV) reversed transcriptive enzyme and human immunodeficiency virus (HIV) reversed transcriptive enzyme and other polypeptide described in WO 98/47921 and its derivative, varient, fragment or mutant and its combination.In yet another embodiment, the RNA enzyme H activity of reversed transcriptive enzyme reduces or reduces in fact, and can be selected from by the following group formed: M-MLV H reversed transcriptive enzyme, RSV H reversed transcriptive enzyme, AMV H reversed transcriptive enzyme, RAV H reversed transcriptive enzyme, MAV H reversed transcriptive enzyme and HIV H reversed transcriptive enzyme and its derivative, varient, fragment or mutant and its combination.Especially concerned reversed transcriptive enzyme comprises AMV RT and M-MLV RT, and optionally RNA enzyme H activity reduces or the AMV RT that reduces in fact and M-MLV RT (such as, AMV RT α H-/BH+ and M-MLV RT H-).The reversed transcriptive enzyme be applicable in the present invention comprises can purchased from the SuperScript of Life Technologies, Inc.
tM, SuperScript
tMiI, ThermoScript
tMand ThermoScript
tMiI.Generally referring to No. 98/47921, PCT publication WO, No. the 5th, 244,797, United States Patent (USP) and the 5th, 668, No. 005, the complete content of each in described patent is incorporated herein by reference.
On the other hand, the invention provides the reaction mixture for polymerization and/or amplification associated nucleic acid sequences (such as, target sequence).In certain embodiments, reaction mixture can comprise detectable label further.Described method can also comprise one or more for detecting detectable label with the quantitative step through amplification of nucleic acid.As used herein, term " detectable label " refers to any one in the various signal transduction molecules of instruction amplification.For example,
green and other DNA binding dye is detectable label.It can be maybe such as nucleic acid intercalating agent or non-intrusive agent that described detectable label can comprise.As used herein, intercalating agent be non-covalently can be inserted into double chain acid molecule stacking base pair between reagent or part.Non-intrusive agent is the reagent be not inserted in double chain acid molecule.Nucleic acid binding agent directly or indirectly can produce detectable signal.Described signal can use such as fluorescence and/or absorbancy directly can detect, or use such as can with detecting by with indirectly can the detecting close to sex any suitable part or part of double-strandednucleic acid, be such as connected to the mark part be substituted or the binding partner of nucleic acid binding agent.Nucleic acid binding agent usually must produce when being attached to double-strandednucleic acid can with when identical reagent in the solution or be attached to single-chain nucleic acid time the signal that the produces detectable signal that distinguishes.For example, the fluorescence that sends when being inserted in double-stranded DNA of the intercalating agent of such as ethidium bromide than be attached to single stranded DNA, RNA or in the solution time strong (referring to such as No. the 5th, 994,056, United States Patent (USP); 6th, 171, No. 785; And/or the 6th, 814, No. 934).Similarly, dactinomycin fluoresces in the RED sector of ultraviolet/visible light spectrum when being attached to single-chain nucleic acid, and fluoresces in the green portion of ultraviolet/visible light spectrum when being attached to double-strandednucleic acid.And in another example, reported photoreactive psoralene 4-aminomethyl-4-5', 8-trimethylpsoralen (AMT) be presented in after being inserted in double-stranded DNA Absorption and fluorescence under long wavelength reduce (people's " photochemistry and photobiology " such as Johnson (Johnson) (
photochem. & Photobiol.), 33:785-791 (1981)).For example, United States Patent (USP) the 4th, 257, No. 774 describe fluorescence intercalating agent (such as, second ingot salt, daunomycin (daunomycin), mepacrine (mepacrine) and acridine orange, 4', 6-diamidino-α-Phenylindole) with the direct combination of DNA.Non-intrusive agent (such as, minor groove binders as described herein, such as Hirst 33258, distamycin (distamycin), T-1384 (netropsin)) also can be applicable.For example, Hirst 33258 (people's " nucleic acids research " such as Se Er (Searle) (
nucl.Acids Res.) 18 (13): 3753-3762 (1990)) show when increasing aim parameter the fluorescence changed to some extent.Minor groove binders in this article other place is described in more detail.
Other DNA binding dye is obtainable for a person skilled in the art and can uses separately or with other reagent of analytical system and/or combination of components.Exemplary DNA binding dye especially can comprise such as acridine (such as, acridine orange, trypaflavine), dactinomycin (people's " J. Mol. BioL " such as Jie En (Jain) (
j.Mol.Biol.) 68:21 (1972)), anthramycin (anthramycin), BOBO
tM-1, BOBO
tM-3, BO-PRO
tM-1, Toyomycin (cbromomycin), DAPI (people's " nucleic acids research " such as Ka Pishensiji (Kapuseinski) (
nucl.Acids res.) 6 (112): 3519 (1979)), daunomycin, distamycin (such as, distamycin D), at United States Patent (USP) the 7th, 387, dyestuff described in No. 887, ellipticine, second ingot salt are (such as, ethidium bromide), fluorine tonka bean camphor (fluorcoumanin), as United States Patent (USP) the 4th, fluorescence intercalating agent described in 257, No. 774,
(blue company (the Cambrex Bio Science Rockland Inc. of Kang Baisi bio-science Roc of Maine State Roc orchid, Rockland, Me.)), Hirst 33258 (Se Er and peace mine-laying (Embrey), " nucleic acids research " (
nucl.Acids Res.) 18:3753-3762 (1990)), Hirst 33342, second phenanthridines (homidium), JO-PRO
tM-1, LIZ dyestuff, LO-PRO
tM-1, mepacrine, mithramycin (mithramycin), NED dyestuff, T-1384,4', 6-diamidino-α-Phenylindole, proflavine (proflavine), POPO
tM-1, POPO
tM-3, PO-PRO
tM-1, propidium iodide, many pyridines ruthenium, S5,
golden,
green I (United States Patent (USP) the 5th, 436, No. 134 and the 5th, 658, No. 751),
green II,
blue,
green,
43,
44,
45,
blue,
11,
13,
15,
16,
20,
23, thiazole orange (the Aldrich Chemical company (Aldrich Chemical Co., Milwaukee, Wis.) of Milwaukee, WI), TOTO
tM-3,
with
(Life Technologies, Inc. of Carlsbad, CA).For example,
green I is (referring to such as No. the 5th, 436,134, United States Patent (USP); 5th, 658, No. 751; And/or the 6th, 569, No. 927) for monitoring PCR reaction.As those skilled in the art will appreciate that, other DNA binding dye also can be suitable.
About purposes as described herein, one or more detectable label and/or quencher can be connected to one or more primer and/or probe (such as, detectable label).Detectable label can transmit when free or when being attached in target nucleic acid one.Detectable label can also transmitting close to during another kind of detectable label.Detectable label can also use to make together with quencher molecule signal only with quencher molecule not enough close to time just can detect.For example, in certain embodiments, analytical system can cause detectable label to discharge from quenching molecules.Can use in some detectable labels any one be marked at primer used and probe in described method herein.As mentioned above, in certain embodiments, detectable label can be connected to the probe that can be incorporated in primer, or can be attached to through amplification target nucleic acid (such as otherwise, can nucleic acid binding agent be detected, such as, insert or non-intrusive dyestuff).When use more than one detectable label, the spectral response curve of each should be different to make described mark to be distinguished from each other, or the signal do not launched separately to make detectable label launch arbitrary detectable label altogether.Exemplary detectable label comprise such as fluorescence dye or fluorophore (such as, can by optical excitation with the chemical group of emitting fluorescence or phosphorescence), can " acceptor dye " etc. of fluorescent signal of quench fluorescence donor dye.Suitable detectable label can comprise such as fluorescein (such as, 5-carboxyl-2,7-dichlorofluorescein, CF (5-FAM), serotonine (5-HAT), 6-JOE, 6-Fluoresceincarboxylic acid (6-FAM), FITC, chloro-2', the 7'-dichlorofluorescein (TET) of 6-carboxyl-Isosorbide-5-Nitrae-two, chloro-2', 4', 5', the 7'-Tetrachlorofluorescein (HEX) of 6-carboxyl-Isosorbide-5-Nitrae-two, chloro-2', the 7'-dimethoxyfluorescein (JOE) of 6-carboxyl-4', 5'-bis-, Alexa
fluorophore (such as, 350,405,430,488,500,514,532,546,555,568,594,610,633,635,647,660,680,700,750),
fluorophore (such as, 492/515, 493/503, 500/510, 505/515, 530/550, 542/563, 558/568, 564/570, 576/589, 581/591, 630/650-X, 650/665-X, 665/676, FL, FL ATP, FI-ceramide, R6G SE, TMR, TMR-X binding substances, TMR-X, SE, TR, TR ATP, TR-X SE), tonka bean camphor (such as, 7-amino-4-methylcoumarin, AMC, AMCA, AMCA-S, AMCA-X, ABQ, CPM methylcoumarin, tonka bean camphor Phalloidine (coumarin phalloidin), Hydroxycoumarin, CMFDA, methoxy coumarin), fluorexon, Calcein-Safranine T, calcein blue, calcium dyestuff (such as, calcium scarlet, calcium is green, calcium orange, white dyes (calcofluor white)), cascade is blue, cascade is yellow, Cy
tMdyestuff (such as, 3, 3.18, 3.5, 5, 5.18, 5.5, 7), cyan GFP, ring-type AMP fluorosensor (FiCRhR), fluorescin (such as, green fluorescent protein (such as, GFP.EGFP), blue fluorescent protein (such as, BFP, EBFP, EBFP2, Azurite, mKalama1), cyan fluorescent protein (such as, ECFP, Cerulean, CyPet), yellow fluorescence protein (such as, YFP, Citrine, Venus, YPet), FRET donor/acceptor is to (such as, fluorescein/tetramethylrhodamine, IAEDANS/ fluorescein, EDANS/dabcyl, fluorescein/fluorescein,
fL, fluorescein/QSY7 and QSY9),
and LysoSensor
tM(such as,
blue DND-22,
blue-white DPX,
yellow HCK-123,
green DND-26,
red DND-99, LysoSensor
tMblue DND-167, LysoSensor
tMgreen DND-189, LysoSensor
tMgreen DND-153, LysoSensor
tMyellow/blue DND-160, LysoSensor
tMyellow/blue 10,000MW dextran), Oregon green (Oregon Green) (such as, 488,488-X, 500,514), rhodamine (rhodamine) (such as, 110, 123, B, B 200, BB, BG, B extra, 5-carboxyl tetramethylrhodamin (5-TAMRA), 5 GLD, 6-carboxyrhodamine 6G, Liz amine (Lissamine), lissamine rhodamine B, Fa Lisiding (Phallicidine), Phalloidine, red, Rhod-2, ROX (6-Carboxy-X-rhodamine), 5-ROX (Carboxy-X-rhodamine), Sulforhodamine B can C, Sulforhodamine G Extra, TAMRA (6-carboxyl tetramethylrhodamin), tetramethylrhodamine (TRITC), WT), texas Red (Texas Red), texas Red-X, VIC and such as described in No. 2009/0197254th, U.S. Patent Application Publication case (mode quoted in full is incorporated herein) other mark, and if those of ordinary skill in the art is by other known mark.If those of ordinary skill in the art is by known, other detectable label (referring to such as No. 2009/0197254th, U.S. Patent Application Publication case (mode quoted in full is incorporated herein)) can also be used.Any one that can use in these systems and detectable label and other systems many and detectable label detects through amplification target nucleic acid.
Some detectable labels can based on sequence (being also referred to as " locus-specific detectable label ") in this article, such as 5'-nuclease probe.Described probe can comprise one or more detectable label.Be known in the art various detectable label, such as described herein
probe is (also referring to United States Patent (USP) the 5th, 538, No. 848 (mode quoted in full is incorporated herein)), various stem-toroidal molecule beacon is (referring to such as United States Patent (USP) the 6th, 103, No. 476 and the 5th, 925, No. 517 and sub-lucky (Tyagi) and carat silent (Kramer), " Nature Biotechnol " (
nature biotechnology) 14:303-308 (1996)), acaulescence or Linear Beacon be (referring to such as No. 99/21881, PCT publication WO; United States Patent (USP) the 6th, 485, No. 901), PNA Molecular Beacons
tM(referring to such as No. the 6th, 355,421, United States Patent (USP) and the 6th, 593, No. 091), linear PNA beacon (referring to people such as such as Ku Bisi towers (Kubista),
sPIE4264:53-58 (2001)), non-FRET probe (referring to such as No. the 6th, 150,097, United States Patent (USP)),
probe (United States Patent (USP) the 6th, 548, No. 250), stem-ring and dual Scorpions
tMprobe (people such as Suo Linasi (Solinas), " nucleic acids research " (
nucleic Acids Research) 29:E96 (2001) and United States Patent (USP) the 6th, 589, No. 743), bulge loop probe (United States Patent (USP) the 6th, 590, No. 091), false knot probe (United States Patent (USP) the 6th, 589, No. 250), cyclicons (United States Patent (USP) the 6th, 383, No. 752), MGB Eclipse
tMprobe (like Bock bio-science), hairpin probe (United States Patent (USP) the 6th, 596, No. 490), peptide nucleic acid(PNA) (PNA) luminescence probe (people's " analytical biochemistry " such as Si Wanweike (Svanvik) (
anal Biochem) 281:26-35 (2001)), self-assembled nanometer particle probe, ferrocene modify probe, such as, described in following each: United States Patent (USP) the 6th, 485, No. 901; Ma Halang adds people such as (Mhlanga), " method " (
methods) 25:463-471 (2001); The people such as Whitcomb (Whitcombe), " Nature Biotechnol " (
nature Biotechnology.) 17:804-807 (1999); The people such as Isaksson (Isacsson), " molecular cell probe " (
molecular Cell Probes.) 14:321-328 (2000); The people such as Si Wanweike (Svanvik), " analytical biochemistry " (
anal Biochem.) 281:26-35 (2000); The people such as Wolf (Wolffs), " biotechnology " (
biotechniques) 766:769-771 (2001); The people such as special soukous (Tsourkas), " nucleic acids research " (
nucleic acids Research.) 30:4208-4215 (2002); The people such as Li Qieli (Riccelli), " nucleic acids research " (
nucleic acids Research) 30:4088-4093 (2002); Open people such as (Zhang), " Acta Biochimica et Biophysica Sinica (Shanghai) " (
acta Biochimica et Biophysica Sinica (Shanghai).) 34:329-332 (2002); The people such as Max Wei Er (Maxwell), " U.S. chemical institute magazine " (J
.Am.Chem.Soc.) 124:9606-9612 (2002); The people such as Blaw moral (Broude), " biotechnology trend " (
trends Biotechnol.) 20:249-56 (2002); People such as yellow (Huang), " chemical toxicology research " (
chem Res.Toxicol.) 15:118-126 (2002); And the people such as remaining (Yu), " U.S. chemical institute magazine " (
j.Am.Chem.Soc.) 14:11155-11161 (2001);
(www.qiagen.com),
(people's " molecular cell probe " such as French (French) (
mol.Cell.Probes) 15:363-374 (2001)), displacement probe (people's " nucleic acids research " such as Lee (Li) (
nucl.Acids Res.) 30:e5 (2002)), hybridization probe (people's " institute of NAS periodical " such as Ka Duluo (Cardullo) (
proc.Natl.Acad.Sci.USA) 85:8790-8794 (1988)), MGB alarm (www.nanogen.com), Q-PNA (take people's " genome research " such as Anda card (Fiandaca) (
genome Res.) 11:609-611 (2001)),
(www.Promega.com), LUX
tMprimer (people's " nucleic acids research " such as Nazarenko (Nazarenko) (
nucleic Acids Res.) 30:e37 (2002)), DzyNA primer (people's " clinical chemistry " such as tod (Todd) (
clin.Chem.) 46:625-630 (2000)).Detectable label can also comprise the undetectable quencher moieties of the fluorescence of quencher detectable label, comprises such as Black Hole Quencher (biological paddy hunter), Iowa
quencher (IDT), QSY quencher (molecular phycobiliprotein complexes) and dimethyl amino-azo-benzene formyl (Dabsyl) and Dabcel sulphonate/manthanoate quencher (love Bock).Detectable label can also comprise two probes, wherein such as fluorophore is on a probe, and quencher is on another probe, wherein two probes hybridize quencher signal together in target, or wherein hybridize in target by change fluorescence change signal characteristic.Illustrative system can also comprise FRET, salicylate/DTPA Fas lignand system (referring to people's " applied chemistry english version " such as such as pool (Oser) difficult to understand (
angew. chem.Int.Engl.) 29 (10): 1167 (1990)), substitution crossing, homologous probe and/or at No. 070685, European patent EP and/or United States Patent (USP) the 6th, 238, the analysis described in No. 927.Detectable label can also comprise fluorescein(e) dye and SO
3instead of the sulfonate derivatives of carboxylate group, the phosphoramidite form of fluorescein, Cy5 phosphoramidite form (can purchased from such as General Electric's Medical Group).The mode that all reference cited above quote in full with it all is hereby incorporated herein.
Composition described herein and method go for the various target nucleic acids of detection and/or quantitative test sample.Target nucleic acid is that design analysis system is to differentiate it or to detect it there is (or not existing) in testing sample neutralization/or carrying out quantitative any nucleic acid to it.Described nucleic acid can comprise such as infectious agent (such as, virus, bacterium, parasite etc.), lysis (such as cancer, diabetes etc.) or for measuring those nucleic acid immunoreactive.Exemplary " test sample " comprises dissimilar sample, biological example sample.Exemplary biological samples comprises such as body fluid (such as, blood, saliva, spinal fluid), tissue sample, food (such as, meat) or beverage (such as, milk) product etc.Such as its expression (or lacking its expression) gene relevant with medical condition of such as transmissible disease (such as, bacterium, virus, fungi, protozoan infection) or cancer can be comprised through express nucleic acid.Method described herein can also be used for the pollutent (such as, bacterium, virus, fungi and/or protozoon) in detection of drugs, food or drink product.Method described herein can also be used for detecting Rare allele when there is wild-type allele (such as, in existence 10
6-10
9a mutant allele in individual wild-type allele situation).Described method is applicable to such as detect minimal residual disease (such as, rare residual cancer cells between the catabasis, especially the sudden change of p53 gene or previous other tumour containment factor gene differentiated in tumour), and/or measure mutational load (frequency of specialized cells sudden change existing in such as healthy tissues (such as blood or urine)).
Any reagent of the change in fluorescence detecting fluorophore or instrument can be used to carry out detection signal.For example, any spectrophotometric thermal cycler can be used to perform detection.The example of spectrophotometric thermal cycler includes, but is not limited to applying biological system (AB)
7000, AB 7300 real-time PCR system, AB 7500 real-time PCR system, AB
7900HT, Bayer Randt (Bio-Rad) ICycler IQ
tM, Xi Feiyide (Cepheid)
iI, Ke Beite study (Corbett Research) Rotor-Gene 3000, Idaho technology (Idaho Technologies) R.A.P.I.D.
tM, MJ studies Chromo 4
tM, Roche applied science (Roche Applied Science)
roche applied science
2.0, Si Tajin (Stratagene) Mx3000P
tMand this tower Tianjin Mx4000
tM.It should be noted that just at fast Development new instrument and any analogous instrument may be used to described method.
Additionally provide the test kit for performing described method herein.As used herein, term " test kit " refers to one group of packaged related component, normally one or more compound or composition.Described test kit can comprise oligonucleotide for being polymerized and/or increase from least one target nucleic acid of sample to, one or more probe marked with detectable label of one or more sanitising agent, nucleic acid polymerase and/or correspondence.Described test kit can also comprise containing the sample for the predefine target nucleic acid in control reaction.Described test kit optionally can also comprise stock solution, damping fluid, enzyme, detectable label or detect required reagent, pipe, film and may be used for the analogue of amplified reaction.In certain embodiments, multiple primer sets is comprised.In one embodiment, described test kit can comprise in following each one or more: such as damping fluid is (such as, Tris), one or more salt (such as, KCl), glycerine, dNTP (dA, dT, dG, dC, dU), restructuring BSA (bovine serum albumin), dyestuff are (such as, ROX inertia reference dye), one or more sanitising agent, polyoxyethylene glycol (PEG), polyvinylpyrrolidone (PVP) and/or gelatin (such as, fish or Niu Yuan).It is to be appreciated that those skilled in the art that other embodiment also containing particular system and test kit.
Described method and composition may be used for the nucleic acid in detection and quantitative sample.Described sample can comprise one or more template and/or one or more target nucleic acid.Described sample can purified or non-purifying.Described sample can be through processing for the biological sample in method provided herein, such as blood, saliva, tear, tissue, urine, ight soil etc.Or if biological sample does not disturb the method provided herein, so it can unprocessed (or not purified) namely use.
example
example 1: by having the amplification restriction of the STAR primer of various overlap length
Design has different overlap length (0,4,5,6,9,11 and 13 Nucleotide) from the forward primer binding site on target sequence and is suppressing the STAR reverse primer of the efficiency in target amplification for assessment of it.Referring to Fig. 3.Two herbicide-tolerant polynucleotides are used: wherein complete the and target sequence and containing between the 6th that hold apart from the 3' in STAR labeled targets district and the 7th base mating (PM) with STAR sequence label completely in STAR region inserts (lin-4) and to miss the target sequence in amplified reaction.Except containing one group of STAR primer of the STAR label mating (PM) completely, be also used in one group of STAR primer that STAR district introduces single base C and A mispairing (MM).With the Power of forward and each 150nM of reverse primer
main mixture adds on real-time PCR instrument (thermal cycle conditions: 95 DEG C/10min, 40 circulation 95 DEG C/15sec and 60 DEG C/1min) in a step and runs PCR in real time.
Exemplaryly to the results are shown in Fig. 4.About the target observations started by 4 Nucleotide overlaps to C
tdisplacement, and MM STAR primer is without impact.When overlap reaches 11 Nucleotide, the amplification of target is suffered significantly to contain, and amplification of missing the target is unaffected.Overlap is increased to 13 Nucleotide and makes target C
tadd 13.4 and make the C that misses the target
tadd 2.7.
example 2: connect background by the containment of STAR primer and improve miRNA and detect.
Workflow shown in Fig. 4 be as submit on November 2nd, 2012 own together, the U.S. Provisional Patent Application the 61/721st of CO-PENDING, the next generation described in No. PCT/USxx/xxxxxth, PCT application that No. 968 and the name submitted to concurrently are therewith called " tiny RNA is caught, detect and quantitatively (Small RNA Capture, Detection and Quantification) "
the example that miRNA analyzes, each mode quoted in full in described application is incorporated herein.In this is analyzed, connect 5' and the 3' end that adapter is connected to miRNA.But, in this Connection Step, adapter-adapter auxiliary connection product can be formed and increase subsequently, producing unwanted background in described analysis.
Design and forward primer contain the overlapping STAR primer of 12 Nucleotide and connect during miRNA analyzes at two end to be tested it.Total serum IgE adds 45 synthesis miRNA and is first connected to 5' with 3' and is connected adapter (being called in Figure 5 " connexon "), then passes through
iII is reverse transcription in RT damping fluid.Circulated in the material through reverse transcription of amplification 1/10th in the main mixture of 1 × pre-amplification of forward and each 250nM of reverse primer by 12 in pre-amplification step.Use standard reverse primer in contrast.Each miRNA target that increases also uses its specificity on Vii7A real-time PCR instrument
analysis detects.Analyze about detection, in the main mixture of 1 × genetic expression, under standard thermal cycle conditions (95 DEG C/10min, 40 circulation 95 DEG C/15sec and 60 DEG C/1min), use the forward of each 900nM and reverse primer and 250nM
probe.Ct value is calculated by automatic baseline and Δ Rn threshold value 0.2.As shown in Figure 6, STAR primer makes the amount of the amplification of the connection adapter through connecting background significantly reduce.Fig. 7 shows and uses STAR primer compared to use reference standard reverse primer, the increase multiple of the detection sensitivity of each miRNA.
example 3: distinguish homology miRNA by STAR primer
Mankind let-7miRNA is very high homology, differs single base between let-7b and let-7c.Use and distinguish let-7b and let-7c from the rest part of family, but the standard primer pair of amplification let-7b and let-7c.As produced miRNA template in example 2 and except using 7900HTS real-time PCR instrument, PCR condition is similar in example 2.Even when the mismatch probe of let-7b, standard analysis still detects let-7b and let-7c (table 1) comparably.For amplified reaction, the cross reactivity of let-7b and let-7c (is calculated as 2 with the STAR primer (Fig. 8) that the STAR sequence label of anti-let-7b designs
Δ Ct (c-b)) from 109% containment to 2% (referring to table 1).Cross reactivity is calculated as 100/2^ Δ Ct (let-7c-let-7b).
Claims (13)
1. an oligonucleotide, it comprises 5' sequence target amplification restricted (STAR) sequence label and target nucleic acid hybridization sequences, wherein said STAR sequence label can described oligonucleotide along described target nucleic acid extension after form stem-ring structure.
2. oligonucleotide according to claim 1, wherein said STAR sequence label comprises all or part of the identical sequence with the target nucleic acid hybridization portion being used from the second primer in amplified reaction with described oligonucleotide one.
3. oligonucleotide according to claim 1, wherein said STAR sequence label with and described oligonucleotide one to be used from all or part of of the second primer in amplified reaction complementary.
4. oligonucleotide according to claim 1, wherein said STAR sequence label comprises a part for the Internal Amplification subsequence of described target nucleic acid.
5. oligonucleotide according to claim 2, wherein said STAR sequence label comprises the Internal Amplification subsequence of all or part of identical sequence with the target nucleic acid hybridization portion of described second primer and described target nucleic acid.
6. oligonucleotide according to claim 3, wherein said STAR sequence label comprises the Internal Amplification subsequence of sequence with all or part of complementation of described second primer and described target nucleic acid.
7. a composition, it comprises the first Oligonucleolide primers and the second Oligonucleolide primers, and wherein said first primer comprises all or part of the identical STAR sequence label with the described target nucleic acid hybridization sequences of described second primer.
8. composition according to claim 8, the length of the sequence wherein shared between described first and described second primer is between about 3 and about 15 Nucleotide.
9. composition according to claim 8, wherein said first primer and described second primer are forward and reverse amplimer pair.
10. a polynucleotide extension products, it is included in the sequence of the STAR sequence label of 5' end and the described STAR sequence label complementation with the 3' side of described STAR sequence label, and wherein said STAR sequence label and described complementary sequence make described extension products can form stem-ring structure.
11. 1 kinds of methods detecting nucleic acid, described method comprises:
Make described target nucleic acid and oligonucleotide hybridization according to claim 1;
Described oligonucleotide is extended to form extension products;
Described extension products is increased to form amplified production; And
Detect amplified production described in presence or absence, detect described target nucleic acid thus.
12. 1 kinds of methods of improper amplification suppressing or block in fact target nucleic acid, described method comprises:
Make described target nucleic acid and oligonucleotide hybridization according to claim 1;
Described oligonucleotide is extended to form extension products;
Described extension products is cultivated together with amplification reaction mixture; And
Detect extension products described in presence or absence.
13. 1 kinds of methods of improper amplification suppressing or block in fact target nucleic acid, described method comprises:
Make described target nucleic acid and oligonucleotide hybridization according to claim 1; With
Described oligonucleotide is extended to form extension products, and wherein said extension products suppresses or reduces in fact the improper amplification of described target nucleic acid.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010205799.3A CN111793621A (en) | 2012-11-02 | 2013-11-04 | Novel compositions, methods and kits for enhancing PCR specificity |
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201261721968P | 2012-11-02 | 2012-11-02 | |
| US61/721,968 | 2012-11-02 | ||
| US201261740242P | 2012-12-20 | 2012-12-20 | |
| US61/740,242 | 2012-12-20 | ||
| PCT/US2013/068335 WO2014071315A1 (en) | 2012-11-02 | 2013-11-04 | Novel compositions, methods and kits for enhancing pcr specificity |
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| WO2014071322A1 (en) | 2014-05-08 |
| CN104955959B (en) | 2020-04-17 |
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